====== CD-HIT User's Guide ======

Last updated: ~~LASTMOD~~

[[http://cd-hit.org]]

Program developed by Weizhong Li's lab at UCSD [[http://weizhongli-lab.org]] and JCVI [[http://jcvi.org]] [[liwz@sdsc.edu]]

===== Introduction =====

CD-HIT is a widely used program for clustering biological sequences to reduce sequence redundancy and improve the performance of other sequence analyses. 

CD-HIT was originally developed to cluster protein sequences to create reference databases with reduced redundancy (Li, et al., 2001) and was then extended to support clustering nucleotide sequences and comparing two datasets (Li and Godzik, 2006). The CD-HIT web server was implemented in 2009, which allows users to cluster or compare sequences without using command-line CD-HIT. The server provides interactive interface and additional visualization tools. 

Currently, CD-HIT package has many programs: cd-hit, cd-hit-2d, cd-hit-est, cd-hit-est-2d, cd-hit-para, cd-hit-2d-para, psi-cd-hit, cd-hit-454, cd-hit-dup, cd-hit-lap, cd-hit-OTU etc. There are also many utility scripts, written in Perl, to help run and analyze CD-HIT jobs. Briefly: 
	
  * cd-hit	Cluster peptide sequences	
  * cd-hit-est	Cluster nucleotide sequences
  * cd-hit-2d	Compare 2 peptide databases	
  * cd-hit-est-2d	Compare 2 nucleotide databases
  * psi-cd-hit	Cluster proteins at <40% cutoff	
  * cd-hit-lap	Identify overlapping reads
  * cd-hit-dup 	Identify duplicates from single or paired Illumina reads	
  * cd-hit-454 	Identify duplicates from 454 reads 
  * cd-hit-otu	Cluster rRNA tags	
  * cd-hit Web server	Cluster user-uploaded data 
  * cd-hit-para	Cluster sequences in parallel on a computer cluster	
  * scripts	Parse results and so on
  * h-cd-hit	Hierarchical clustering 		

Recently development of cd-hit, especially the multiple-threaded version introduced in 2012 (Fu et al), enables clustering of very large NGS datasets. For example, it only took cd-hit less than a day on a 32-core computer to cluster a few hundred million protein sequences from a metagenomics study. 

===== Algorithm =====
__**[You can skip this session if you are eager to use the programs, no problem].**__

Algorithms for CD-HIT were described in these papers.

  *1. Weizhong Li, Lukasz Jaroszewski & Adam Godzik. Clustering of highly homologous sequences to reduce the size of large protein databases. Bioinformatics (2001) 17:282-283, [[http://bioinformatics.oupjournals.org/cgi/reprint/17/3/282.pdf|PDF]], [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11294794|Pubmed]]
  *2. Weizhong Li, Lukasz Jaroszewski & Adam Godzik. Tolerating some redundancy significantly speeds up clustering of large protein databases. Bioinformatics (2002) 18: 77-82, [[http://bioinformatics.oupjournals.org/cgi/reprint/18/1/77.pdf|PDF]], [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11836214|Pubmed]]
  *3. Weizhong Li & Adam Godzik. Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics (2006) 22:1658-1659 [[http://bioinformatics.oxfordjournals.org/cgi/reprint/22/13/1658|PDF]], [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=Abstract&list_uids=16731699|Pubmed]]
  *4. Limin Fu, Beifang Niu, Zhengwei Zhu, Sitao Wu and Weizhong Li. CD-HIT: accelerated for clustering the next generation sequencing data. Bioinformatics (2012) 28:3150-3152, doi: 10.1093/bioinformatics/bts565 [[http://bioinformatics.oxfordjournals.org/content/28/23/3150|PDF]]
  *5. Ying Huang, Beifang Niu, Ying Gao, Limin Fu and Weizhong Li. CD-HIT Suite: a web server for clustering and comparing biological sequences. Bioinformatics, (2010). 26:680 [[http://bioinformatics.oxfordjournals.org/cgi/reprint/btq003v1|PDF]] [[http://www.ncbi.nlm.nih.gov/pubmed/20053844|Pubmed]]
  *6. Beifang Niu, Limin Fu, Shulei Sun and Weizhong Li, Artificial and natural duplicates in pyrosequencing reads of metagenomic data. BMC Bioinformatics, (2010) 11:187 [[http://www.biomedcentral.com/content/pdf/1471-2105-11-187.pdf|PDF]] [[http://www.ncbi.nlm.nih.gov/pubmed/20388221|Pubmed]]
  *7. Weizhong Li, Limin Fu, Beifang Niu, Sitao Wu and John Wooley. Ultrafast clustering algorithms for metagenomic sequence analysis. Briefings in Bioinformatics, (2012) 13 (6): 656-668. doi: 10.1093/bib/bbs035 [[http://bib.oxfordjournals.org/content/13/6/656|PDF]]  


==== CD-HIT clustering algorithm ====

Clustering a sequence database usually requires all-by-all comparisons; therefore it is very time-consuming. Many methods use BLAST to compute the all vs. all similarities. It is very difficult for these methods to cluster large databases. While CD-HIT can avoid many pairwise sequence alignments with a short word based heuristics. 

CD-HIT is a greedy incremental clustering approach. The basic CD-HIT algorithm sorts the input sequences from long to short, and processes them sequentially from the longest to the shortest. The first sequence is automatically classified as the first cluster representative sequence. Then each query sequence of the remaining sequences is compared to the representative sequences found before it, and is classified as redundant or representative based on whether it is similar to one of the existing representative sequences. In default manner (fast mode), a query is grouped into the first representative without comparing to other representatives. In accurate mode, a query is compared to all representatives and grouped to the most similar one.

Based on this greedy method, we established several integrated heuristics that make CD-HIT very efficient.

**Index table**: Most sequence alignment tools use short words or k-mer (k is the length) to speed up computation. For example, BLASTN uses 11-mer and BLASTP uses 3-mer by default. An index table, as opposite to a hash table used in most fast alignment methods, uses a unique index for every unique k-mer, therefore an index table is much faster than a hash table. In CD-HIT, we use k=2~5 for proteins and k=8~12 for DNAs, because the all the k-mers can be indexed in computer memory. 

**Short word filter**: Two sequences of a certain identity must share at least a specific number of identical k-mers. It is possible to tell that the identity between two sequences is below a cutoff by counting common k-mers. The filter checks the common k-mers and rejects unnecessary alignments.

**Short word statistics**: It is the key for the high efficiency of a short word filter (Li, et al., 2002). Through statistical analysis of real alignments, we identified the distribution of common k-mer at different sequence lengths and identities, and applied the results to short word filter.

**Banded alignment**: The short word filter not only filters out unnecessary alignment, when alignment is needed, it also identifies a narrow band for banded dynamic programming alignment, which is much faster than regular dynamic programming.

**Reduced alphabet (to be implemented)**: This is for protein clustering. In reduced alphabet, a group of exchangeable residues are reduced to a single residue (I/V/L==>I, S/T==>S, D/E==>D, K/R==>K, F/Y==>F), and then conservative mutations would appear as identities in sequence alignments. It improves the short word filter for clustering at low sequence identity below 50%.

**Gapped word (to be implemented)**: Short word filter using gapped word allows mismatch within a word such as âACEâ vs âAMEâ, âACFEâ vs âAMYEâ, and âAACTTâ vs âAAGTTâ, which can be written as â101â, â1001â and â11011â. At low identity cutoff, a gapped word is more efficient than an ungapped word for filtering.



==== Algorithm limitations ====

A limitation of short word filter is that it can not be used below certain clustering thresholds. 
For proteins:
  * word size 5 is for thresholds 0.7 ~ 1.0
  * word size 4 is for thresholds 0.6 ~ 0.7
  * word size 3 is for thresholds 0.5 ~ 0.6
  * word size 2 is for thresholds 0.4 ~ 0.5 (also see psi-cd-hit)

For DNAs:
  * Word size 10-11 is for thresholds 0.95 ~ 1.0
  * Word size 8,9 is for thresholds 0.90 ~ 0.95
  * Word size 7 is for thresholds 0.88 ~ 0.9
  * Word size 6 is for thresholds 0.85 ~ 0.88
  * Word size 5 is for thresholds 0.80 ~ 0.85
  * Word size 4 is for thresholds 0.75 ~ 0.8

Because of the algorithm, cd-hit may not be used for clustering proteins at <40% identity. Cd-hit-est cannot cluster very long sequences either (e.g. genome sized sequences). In such cases, please use PSI-CD-HIT, which will be introduced in following sections.



 
===== User's Guide =====

==== Installation ==== 

It can be copied under the GNU General Public License version 2 (GPLv2).

Most CD-HIT programs were written in C++. Installing CD-HIT package is very simple:
  * download current CD-HIT at [[https://github.com/weizhongli/cdhit/releases]], for example cd-hit-v4.6.6-2016-0711.tar.gz
  * unpack the file with " tar xvf cd-hit-v4.6.6-2016-0711.tar.gz --gunzip"
  * change dir by "cd cd-hit-v4.6.6-2016-0711"
  * compile the programs by "make" with multi-threading (default), or by "make openmp=no" without multi-threading (on old systems without OpenMP)
  * cd cd-hit-auxtools
  * compile cd-hit-auxtools by "make"

==== CD-HIT ====

CD-HIT clusters proteins into clusters that meet a user-defined similarity threshold, usually a sequence identity. Each cluster has one representative sequence. The input is a protein dataset in fasta format and the output are two files: a fasta file of representative sequences and a text file of list of clusters.

Basic command:
  cd-hit -i nr -o nr100 -c 1.00 -n 5 -M 16000 âd 0 -T 8
  cd-hit -i db -o db90 -c 0.9 -n 5 -M 16000 âd 0 -T 8, 

where\\ 
''db'' is the filename of input, \\
''db90'' is output, \\
''-c 1.0'', means 100% identity, is the clustering threshold\\
''-c 0.9'', means 90% identity, is the clustering threshold\\
''-n 5'' is the word size\\
''-d 0'' use sequence name in fasta header till the first white space\\
''-M 16000'', to use 16GB RAM\\
''-T 8'', to use 8 threads\\

Choose of word size:

''-n 5'' for thresholds 0.7 ~ 1.0\\
''-n 4'' for thresholds 0.6 ~ 0.7\\
''-n 3'' for thresholds 0.5 ~ 0.6\\
''-n 2'' for thresholds 0.4 ~ 0.5\\


Complete options:

__**The most updated options are available from the command line version of the programs. Running the programs without any argument will print out the detailed options.**__

<code>
   -i	input filename in fasta format, required
   -o	output filename, required
   -c	sequence identity threshold, default 0.9
 	this is the default cd-hit's "global sequence identity" calculated as:
 	number of identical amino acids in alignment
 	divided by the full length of the shorter sequence
   -G	use global sequence identity, default 1
 	if set to 0, then use local sequence identity, calculated as :
 	number of identical amino acids in alignment
 	divided by the length of the alignment
 	NOTE!!! don't use -G 0 unless you use alignment coverage controls
 	see options -aL, -AL, -aS, -AS
   -b	band_width of alignment, default 20
   -M	memory limit (in MB) for the program, default 800; 0 for unlimitted;
   -T	number of threads, default 1; with 0, all CPUs will be used
   -n	word_length, default 5, see user's guide for choosing it
   -l	length of throw_away_sequences, default 10
   -t	tolerance for redundance, default 2
   -d	length of description in .clstr file, default 20
 	if set to 0, it takes the fasta defline and stops at first space
   -s	length difference cutoff, default 0.0
 	if set to 0.9, the shorter sequences need to be
 	at least 90% length of the representative of the cluster
   -S	length difference cutoff in amino acid, default 999999
 	if set to 60, the length difference between the shorter sequences
 	and the representative of the cluster can not be bigger than 60
   -aL	alignment coverage for the longer sequence, default 0.0
 	if set to 0.9, the alignment must covers 90% of the sequence
   -AL	alignment coverage control for the longer sequence, default 99999999
 	if set to 60, and the length of the sequence is 400,
 	then the alignment must be >= 340 (400-60) residues
   -aS	alignment coverage for the shorter sequence, default 0.0
 	if set to 0.9, the alignment must covers 90% of the sequence
   -AS	alignment coverage control for the shorter sequence, default 99999999
 	if set to 60, and the length of the sequence is 400,
 	then the alignment must be >= 340 (400-60) residues
   -A	minimal alignment coverage control for the both sequences, default 0
 	alignment must cover >= this value for both sequences 
   -uL	maximum unmatched percentage for the longer sequence, default 1.0
 	if set to 0.1, the unmatched region (excluding leading and tailing gaps)
 	must not be more than 10% of the sequence
   -uS	maximum unmatched percentage for the shorter sequence, default 1.0
 	if set to 0.1, the unmatched region (excluding leading and tailing gaps)
 	must not be more than 10% of the sequence
   -U	maximum unmatched length, default 99999999
 	if set to 10, the unmatched region (excluding leading and tailing gaps)
 	must not be more than 10 bases
   -B	1 or 0, default 0, by default, sequences are stored in RAM
 	if set to 1, sequence are stored on hard drive
 	!! No longer supported !!
   -p	1 or 0, default 0
 	if set to 1, print alignment overlap in .clstr file
   -g	1 or 0, default 0
 	by cd-hit's default algorithm, a sequence is clustered to the first 
 	cluster that meet the threshold (fast cluster). If set to 1, the program
 	will cluster it into the most similar cluster that meet the threshold
 	(accurate but slow mode)
 	but either 1 or 0 won't change the representatives of final clusters
   -sc	sort clusters by size (number of sequences), default 0, output clusters by decreasing length
 	if set to 1, output clusters by decreasing size
   -sf	sort fasta/fastq by cluster size (number of sequences), default 0, no sorting
 	if set to 1, output sequences by decreasing cluster size
   -bak	write backup cluster file (1 or 0, default 0)
   -h	print this help

</code>

Alignment coverage control:

See the figure below, the -aL, -AL, -aS and -AS options can be used to specify the alignment coverage on both the representative sequence and other sequences. -s and -S can control the length difference between the representative sequence and other sequences.

{{ :cd-hit-figure2.png }}


''
aL = R<sub>a</sub> / R\\
AL = R - R<sub>a</sub>\\
aS = S<sub>a</sub> / S\\
AS = S - S<sub>a</sub>\\
s = S<sub>a</sub> / R<sub>a</sub>\\
S = R / S\\
U = S<sub>1</sub> + S<sub>2</sub>\\
uL = U / R\\
uS = U / S
''

Output:

The output .clstr file looks like
<code>
>Cluster 0
0 2799aa, >PF04998.6|RPOC2_CHLRE/275-3073... *
>Cluster 1
0 2214aa, >PF06317.1|Q6Y625_9VIRU/1-2214... at 80%
1 2215aa, >PF06317.1|O09705_9VIRU/1-2215... at 84%
2 2217aa, >PF06317.1|Q6Y630_9VIRU/1-2217... *
3 2216aa, >PF06317.1|Q6GWS6_9VIRU/1-2216... at 84%
4 527aa, >PF06317.1|Q67E14_9VIRU/6-532... at 63%
>Cluster 2
0 2202aa, >PF06317.1|Q6UY61_9VIRU/8-2209... at 60%
1 2208aa, >PF06317.1|Q6IVU4_JUNIN/1-2208... *
2 2207aa, >PF06317.1|Q6IVU0_MACHU/1-2207... at 73%
3 2208aa, >PF06317.1|RRPO_TACV/1-2208... at 69%
</code>
where\\
a ">" starts a new cluster\\
a "*" at the end means that this sequence is the representative of this cluster\\
a "%" is the identity between this sequence and the representative

==== CD-HIT-2D ====

CD-HIT-2D compares 2 protein datasets (db1, db2). It identifies the sequences in db2 that are similar to db1 at a certain threshold. The input are two protein datasets (db1, db2) in fasta format and the output are two files: a fasta file of proteins in db2 that are not similar to db1 and a text file that lists similar sequences between db1 & db2.

Basic command:
  cd-hit-2d -i db1 -i2 db2 -o db2novel -c 0.9 -n 5 -d 0 -M 16000 -T 8
where\\
''db1'' & ''db2'' are inputs,\\
''db2novel'' is output,\\
''0.9'' means 90% identity, is the comparing threshold\\
''5'' is the size of word

Please note that by default, cd-hit only lists matches where sequences in db2 are
not longer than sequences in db1. You may use options -S2 or -s2 to overwrite
this default. You can also swap db1 and db2:

  cd-hit-2d -i db1 -i2 db2 -o db2novel -c 0.9 -n 5 -d 0 -M 16000 -T 8 -s2 0.9
  cd-hit-2d -i db2 -i2 db1 -o db1novel -c 0.9 -n 5 -d 0 -M 16000 -T 8 (swap db1 and db2)

Choose of word size (same as cd-hit):
<code>
-n 5 for thresholds 0.7 ~ 1.0
-n 4 for thresholds 0.6 ~ 0.7
-n 3 for thresholds 0.5 ~ 0.6
-n 2 for thresholds 0.4 ~ 0.5 
</code>

Options:
<code>
   -i	input filename for db1 in fasta format, required
   -i2	input filename for db2 in fasta format, required
   -o	output filename, required
   -c	sequence identity threshold, default 0.9
 	this is the default cd-hit's "global sequence identity" calculated as:
 	number of identical amino acids in alignment
 	divided by the full length of the shorter sequence
   -G	use global sequence identity, default 1
 	if set to 0, then use local sequence identity, calculated as :
 	number of identical amino acids in alignment
 	divided by the length of the alignment
 	NOTE!!! don't use -G 0 unless you use alignment coverage controls
 	see options -aL, -AL, -aS, -AS
   -b	band_width of alignment, default 20
   -M	memory limit (in MB) for the program, default 800; 0 for unlimitted;
   -T	number of threads, default 1; with 0, all CPUs will be used
   -n	word_length, default 5, see user's guide for choosing it
   -l	length of throw_away_sequences, default 10
   -t	tolerance for redundance, default 2
   -d	length of description in .clstr file, default 20
 	if set to 0, it takes the fasta defline and stops at first space
   -s	length difference cutoff, default 0.0
 	if set to 0.9, the shorter sequences need to be
 	at least 90% length of the representative of the cluster
   -S	length difference cutoff in amino acid, default 999999
 	if set to 60, the length difference between the shorter sequences
 	and the representative of the cluster can not be bigger than 60
   -s2	length difference cutoff for db1, default 1.0
 	by default, seqs in db1 >= seqs in db2 in a same cluster
 	if set to 0.9, seqs in db1 may just >= 90% seqs in db2
   -S2	length difference cutoff, default 0
 	by default, seqs in db1 >= seqs in db2 in a same cluster
 	if set to 60, seqs in db2 may 60aa longer than seqs in db1
   -aL	alignment coverage for the longer sequence, default 0.0
 	if set to 0.9, the alignment must covers 90% of the sequence
   -AL	alignment coverage control for the longer sequence, default 99999999
 	if set to 60, and the length of the sequence is 400,
 	then the alignment must be >= 340 (400-60) residues
   -aS	alignment coverage for the shorter sequence, default 0.0
 	if set to 0.9, the alignment must covers 90% of the sequence
   -AS	alignment coverage control for the shorter sequence, default 99999999
 	if set to 60, and the length of the sequence is 400,
 	then the alignment must be >= 340 (400-60) residues
   -A	minimal alignment coverage control for the both sequences, default 0
 	alignment must cover >= this value for both sequences 
   -uL	maximum unmatched percentage for the longer sequence, default 1.0
 	if set to 0.1, the unmatched region (excluding leading and tailing gaps)
 	must not be more than 10% of the sequence
   -uS	maximum unmatched percentage for the shorter sequence, default 1.0
 	if set to 0.1, the unmatched region (excluding leading and tailing gaps)
 	must not be more than 10% of the sequence
   -U	maximum unmatched length, default 99999999
 	if set to 10, the unmatched region (excluding leading and tailing gaps)
 	must not be more than 10 bases
   -B	1 or 0, default 0, by default, sequences are stored in RAM
 	if set to 1, sequence are stored on hard drive
 	!! No longer supported !!
   -p	1 or 0, default 0
 	if set to 1, print alignment overlap in .clstr file
   -g	1 or 0, default 0
 	by cd-hit's default algorithm, a sequence is clustered to the first 
 	cluster that meet the threshold (fast cluster). If set to 1, the program
 	will cluster it into the most similar cluster that meet the threshold
 	(accurate but slow mode)
 	but either 1 or 0 won't change the representatives of final clusters
   -bak	write backup cluster file (1 or 0, default 0)
   -h	print this help

</code>

==== CD-HIT-EST ====

CD-HIT-EST clusters a nucleotide dataset into clusters that meet a
user-defined similarity threshold, usually a sequence identity. The input is
a DNA/RNA dataset in fasta format and the output are two files: a fasta file
of representative sequences and a text file of list of clusters. 
Since eukaryotic genes usually have long introns, which cause long gaps, it is
difficult to make full-length alignments for these genes. So, CD-HIT-EST is
good for non-intron containing sequences like EST.  

Basic command:
  cd-hit-est -i est_human -o est_human95 -c 0.95 -n 10 -d 0 -M 16000 - T 8
  cd-hit-est -i R1.fa -j R2.fa -o R1.95.fa -op R2.95.fa -P 1 -c 0.95 -n 10 -d 0 -M 16000 - T 8   

Choose of word size:
<code>
-n 10, 11 for thresholds 0.95 ~ 1.0
-n 8,9    for thresholds 0.90 ~ 0.95
-n 7      for thresholds 0.88 ~ 0.9
-n 6      for thresholds 0.85 ~ 0.88
-n 5      for thresholds 0.80 ~ 0.85
-n 4      for thresholds 0.75 ~ 0.8 
</code>

Options:
<code>
   -i	input filename in fasta format, required
   -j	input filename in fasta/fastq format for R2 reads if input are paired end (PE) files
 	 -i R1.fq -j R2.fq -o output_R1 -op output_R2 or
 	 -i R1.fa -j R2.fa -o output_R1 -op output_R2 
   -o	output filename, required
   -op	output filename for R2 reads if input are paired end (PE) files
   -c	sequence identity threshold, default 0.9
 	this is the default cd-hit's "global sequence identity" calculated as:
 	number of identical amino acids in alignment
 	divided by the full length of the shorter sequence
   -G	use global sequence identity, default 1
 	if set to 0, then use local sequence identity, calculated as :
 	number of identical amino acids in alignment
 	divided by the length of the alignment
 	NOTE!!! don't use -G 0 unless you use alignment coverage controls
 	see options -aL, -AL, -aS, -AS
   -b	band_width of alignment, default 20
   -M	memory limit (in MB) for the program, default 800; 0 for unlimitted;
   -T	number of threads, default 1; with 0, all CPUs will be used
   -n	word_length, default 10, see user's guide for choosing it
   -l	length of throw_away_sequences, default 10
   -d	length of description in .clstr file, default 20
 	if set to 0, it takes the fasta defline and stops at first space
   -s	length difference cutoff, default 0.0
 	if set to 0.9, the shorter sequences need to be
 	at least 90% length of the representative of the cluster
   -S	length difference cutoff in amino acid, default 999999
 	if set to 60, the length difference between the shorter sequences
 	and the representative of the cluster can not be bigger than 60
   -aL	alignment coverage for the longer sequence, default 0.0
 	if set to 0.9, the alignment must covers 90% of the sequence
   -AL	alignment coverage control for the longer sequence, default 99999999
 	if set to 60, and the length of the sequence is 400,
 	then the alignment must be >= 340 (400-60) residues
   -aS	alignment coverage for the shorter sequence, default 0.0
 	if set to 0.9, the alignment must covers 90% of the sequence
   -AS	alignment coverage control for the shorter sequence, default 99999999
 	if set to 60, and the length of the sequence is 400,
 	then the alignment must be >= 340 (400-60) residues
   -A	minimal alignment coverage control for the both sequences, default 0
 	alignment must cover >= this value for both sequences 
   -uL	maximum unmatched percentage for the longer sequence, default 1.0
 	if set to 0.1, the unmatched region (excluding leading and tailing gaps)
 	must not be more than 10% of the sequence
   -uS	maximum unmatched percentage for the shorter sequence, default 1.0
 	if set to 0.1, the unmatched region (excluding leading and tailing gaps)
 	must not be more than 10% of the sequence
   -U	maximum unmatched length, default 99999999
 	if set to 10, the unmatched region (excluding leading and tailing gaps)
 	must not be more than 10 bases
   -B	1 or 0, default 0, by default, sequences are stored in RAM
 	if set to 1, sequence are stored on hard drive
 	!! No longer supported !!
   -P	input paired end (PE) reads, default 0, single file
 	if set to 1, please use -i R1 -j R2 to input both PE files
   -cx	length to keep after trimming the tail of sequence, default 0, not trimming
 	if set to 50, the program only uses the first 50 letters of input sequence
   -cy	length to keep after trimming the tail of R2 sequence, default 0, not trimming
 	if set to 50, the program only uses the first 50 letters of input R2 sequence
 	e.g. -cx 100 -cy 80 for paired end reads
   -ap	alignment position constrains,  default 0, no constrain
 	if set to 1, the program will force sequences to align at beginings
 	when set to 1, the program only does +/+ alignment
   -p	1 or 0, default 0
 	if set to 1, print alignment overlap in .clstr file
   -g	1 or 0, default 0
 	by cd-hit's default algorithm, a sequence is clustered to the first 
 	cluster that meet the threshold (fast cluster). If set to 1, the program
 	will cluster it into the most similar cluster that meet the threshold
 	(accurate but slow mode)
 	but either 1 or 0 won't change the representatives of final clusters
   -r	1 or 0, default 1, by default do both +/+ & +/- alignments
 	if set to 0, only +/+ strand alignment
   -mask	masking letters (e.g. -mask NX, to mask out both 'N' and 'X')
   -match	matching score, default 2 (1 for T-U and N-N)
   -mismatch	mismatching score, default -2
   -gap	gap opening score, default -6
   -gap-ext	gap extension score, default -1
   -bak	write backup cluster file (1 or 0, default 0)
   -sc	sort clusters by size (number of sequences), default 0, output clusters by decreasing length
 	if set to 1, output clusters by decreasing size
   -sf	sort fasta/fastq by cluster size (number of sequences), default 0, no sorting
 	if set to 1, output sequences by decreasing cluster size
   -h	print this help


</code>

==== CD-HIT-EST-2D ====

CD-HIT-EST-2D compares 2 nucleotide datasets (db1, db2). It identifies the
sequences in db2 that are similar to db1 at a certain threshold. The input are
two DNA/RNA datasets (db1, db2) in fasta format and the output are two files:
a fasta file of sequences in db2 that are not similar to db1 and a text file
that lists similar sequences between db1 & db2.  
For same reason as CD-HIT-EST, CD-HIT-EST-2D is good for non-intron containing
sequences like EST.  

Basic command:
  cd-hit-est-2d -i mrna_human -i2 est_human -o est_human_novel -c 0.95 -n 10 -d 0 -M 16000 - T 8
  cd-hit-est-2d -i db1.R1.fa -j db1.R2.fa -i2 db2.R1.fa -j2 db2.R2.fa -o db2_novel.R1.fa -op db2_novel.R2.fa -P 1 -c 0.95 -n 10 -d 0 -M 16000 - T 8
    
Choose of word size and options are the same as CD-HIT-EST:

Options:
<code>
   -i	input filename for db1 in fasta format, required
   -i2	input filename for db2 in fasta format, required
   -j, -j2	input filename in fasta/fastq format for R2 reads if input are paired end (PE) files
 	 -i db1-R1.fq -j db1-R2.fq -i2 db2-R1.fq -j2 db2-R2.fq -o output_R1 -op output_R2 or
 	 -i db1-R1.fa -j db1-R2.fa -i2 db2-R1.fq -j2 db2-R2.fq -o output_R1 -op output_R2 
   -o	output filename, required
   -op	output filename for R2 reads if input are paired end (PE) files
   -c	sequence identity threshold, default 0.9
 	this is the default cd-hit's "global sequence identity" calculated as:
 	number of identical amino acids in alignment
 	divided by the full length of the shorter sequence
   -G	use global sequence identity, default 1
 	if set to 0, then use local sequence identity, calculated as :
 	number of identical amino acids in alignment
 	divided by the length of the alignment
 	NOTE!!! don't use -G 0 unless you use alignment coverage controls
 	see options -aL, -AL, -aS, -AS
   -b	band_width of alignment, default 20
   -M	memory limit (in MB) for the program, default 800; 0 for unlimitted;
   -T	number of threads, default 1; with 0, all CPUs will be used
   -n	word_length, default 10, see user's guide for choosing it
   -l	length of throw_away_sequences, default 10
   -d	length of description in .clstr file, default 20
 	if set to 0, it takes the fasta defline and stops at first space
   -s	length difference cutoff, default 0.0
 	if set to 0.9, the shorter sequences need to be
 	at least 90% length of the representative of the cluster
   -S	length difference cutoff in amino acid, default 999999
 	if set to 60, the length difference between the shorter sequences
 	and the representative of the cluster can not be bigger than 60
   -s2	length difference cutoff for db1, default 1.0
 	by default, seqs in db1 >= seqs in db2 in a same cluster
 	if set to 0.9, seqs in db1 may just >= 90% seqs in db2
   -S2	length difference cutoff, default 0
 	by default, seqs in db1 >= seqs in db2 in a same cluster
 	if set to 60, seqs in db2 may 60aa longer than seqs in db1
   -aL	alignment coverage for the longer sequence, default 0.0
 	if set to 0.9, the alignment must covers 90% of the sequence
   -AL	alignment coverage control for the longer sequence, default 99999999
 	if set to 60, and the length of the sequence is 400,
 	then the alignment must be >= 340 (400-60) residues
   -aS	alignment coverage for the shorter sequence, default 0.0
 	if set to 0.9, the alignment must covers 90% of the sequence
   -AS	alignment coverage control for the shorter sequence, default 99999999
 	if set to 60, and the length of the sequence is 400,
 	then the alignment must be >= 340 (400-60) residues
   -A	minimal alignment coverage control for the both sequences, default 0
 	alignment must cover >= this value for both sequences 
   -uL	maximum unmatched percentage for the longer sequence, default 1.0
 	if set to 0.1, the unmatched region (excluding leading and tailing gaps)
 	must not be more than 10% of the sequence
   -uS	maximum unmatched percentage for the shorter sequence, default 1.0
 	if set to 0.1, the unmatched region (excluding leading and tailing gaps)
 	must not be more than 10% of the sequence
   -U	maximum unmatched length, default 99999999
 	if set to 10, the unmatched region (excluding leading and tailing gaps)
 	must not be more than 10 bases
   -B	1 or 0, default 0, by default, sequences are stored in RAM
 	if set to 1, sequence are stored on hard drive
 	!! No longer supported !!
   -P	input paired end (PE) reads, default 0, single file
 	if set to 1, please use -i R1 -j R2 to input both PE files
   -cx	length to keep after trimming the tail of sequence, default 0, not trimming
 	if set to 50, the program only uses the first 50 letters of input sequence
   -cy	length to keep after trimming the tail of R2 sequence, default 0, not trimming
 	if set to 50, the program only uses the first 50 letters of input R2 sequence
 	e.g. -cx 100 -cy 80 for paired end reads
   -p	1 or 0, default 0
 	if set to 1, print alignment overlap in .clstr file
   -g	1 or 0, default 0
 	by cd-hit's default algorithm, a sequence is clustered to the first 
 	cluster that meet the threshold (fast cluster). If set to 1, the program
 	will cluster it into the most similar cluster that meet the threshold
 	(accurate but slow mode)
 	but either 1 or 0 won't change the representatives of final clusters
   -r	1 or 0, default 1, by default do both +/+ & +/- alignments
 	if set to 0, only +/+ strand alignment
   -mask	masking letters (e.g. -mask NX, to mask out both 'N' and 'X')
   -match	matching score, default 2 (1 for T-U and N-N)
   -mismatch	mismatching score, default -2
   -gap	gap opening score, default -6
   -gap-ext	gap extension score, default -1
   -bak	write backup cluster file (1 or 0, default 0)
   -h	print this help

</code>


==== CD-HIT-454 clustering ====

We implemented a program called cd-hit-454 to identify duplicated 454 reads by reengineering cd-hit-est. Duplicates are either exactly identical or meet these criteria includes: (1) they start at the same position; (2) their lengths can be different, but shorter one must be fully aligned with the longer one (the seed); (3) they can only have 4% mismatches (insertion, deletion, and substitution); and (4) only 1 base is allowed per insertion or deletion. Here, (3) and (4) can be adjusted by users. We allow mismatches in order to tolerate sequencing errors. To find duplicates in Illumina reads, please use cd-hit-dup (see later sections)

Basic command:
  cd-hit-454 -i 454_reads -o 454_reads_95 -c 0.95 -n 10 -d 0 -M 16000 - T 8  

Options:
<code>
   -i	input filename in fasta format, required
   -o	output filename, required
   -c	sequence identity threshold, default 0.98
 	this is a "global sequence identity" calculated as :
 	number of identical amino acids in alignment
 	divided by the full length of the shorter sequence + gaps
   -b	band_width of alignment, default 10
   -M	memory limit (in MB) for the program, default 800; 0 for unlimitted;
   -T	number of threads, default 1; with 0, all CPUs will be used
   -n	word_length, default 10, see user's guide for choosing it
   -aL	alignment coverage for the longer sequence, default 0.0
 	if set to 0.9, the alignment must covers 90% of the sequence
   -AL	alignment coverage control for the longer sequence, default 99999999
 	if set to 60, and the length of the sequence is 400,
 	then the alignment must be >= 340 (400-60) residues
   -aS	alignment coverage for the shorter sequence, default 0.0
 	if set to 0.9, the alignment must covers 90% of the sequence
   -AS	alignment coverage control for the shorter sequence, default 99999999
 	if set to 60, and the length of the sequence is 400,
 	then the alignment must be >= 340 (400-60) residues
   -B	1 or 0, default 0, by default, sequences are stored in RAM
 	if set to 1, sequence are stored on hard drive
 	it is recommended to use -B 1 for huge databases
   -g	1 or 0, default 0
 	by cd-hit's default algorithm, a sequence is clustered to the first 
 	cluster that meet the threshold (fast cluster). If set to 1, the program
 	will cluster it into the most similar cluster that meet the threshold
 	(accurate but slow mode)
 	but either 1 or 0 won't change the representatives of final clusters
   -D	max size per indel, default 1
   -match	matching score, default 2
   -mismatch	mismatching score, default -1
   -gap	gap opening score, default -3
   -gap-ext	gap extension score, default -1
   -bak	write backup cluster file (1 or 0, default 0)
</code>

==== Multi-threaded programs ====

Multi-threaded cd-hit programs were implemented with OpenMP. Option "-T
n" will enable cd-hit to run in parallel in a single multi-core computer.
The default value of n is 1 (single thread). "-T 0" will use all the
cores in that computer. We have run cd-hit on 4-core, 8-core to 16-core
computers and have observed a great speedup.

==== CD-HIT-PARA ====

__** [CD-HIT-PARA is no longer supported, since the multi-threaded cd-hit become available.] **__

CD-HIT-PARA is a script that runs cd-hit, cd-hit-est in a parallel mode. It
splits the input database; runs cd-hit or cd-hit-est in parallel on a computer
cluster; and finally merges the outputs into a single file.  You can run it as
you run cd-hit or cd-hit-est. The input is a protein or DNA/RNA dataset in
fasta format and the output are two files: a fasta file of representative
sequences and a text file of list of clusters. 

There are two ways to run jobs on a cluster: by ssh to a remote computer and
by queuing system (PBS and SGE are implemented). In any case, you should have
a shared file system, the path to your working directory must be same on all
the remote computers. 

This script can also be used if you are clustering a very large database and
your computer doesn't have enough RAM. In that case, all the divided jobs
will still run on a single computer. 

Implementation (see figure below)

  - divide input db into many small dbs in decreasing length
  - clusters the 1st db by cd-hit
  - run cd-hit-2d for other dbs against 1st db
  - repeat cd-hit and cd-hit-2d runs till done
  - Combine the results

{{ :cd-hit-figure3.png }}

Basic command:
  cd-hit-para.pl -i nr90 -o nr60 -c 0.6 -n 4 --B hosts --S 64
  
where\\
''--B'' hosts is a file with available hostnames\\
''--S'' 64 is the number to split input db into, this number should be several times the number of hosts 

More options:
<code>
--P program, "cd-hit" or "cd-hit-est", default "cd-hit"
--B filename of list of hosts,
    requred unless -Q or -L option is supplied
--L number of cpus on local computer, default 0
    when you are not running it over a cluster, you can use
    this option to divide a big clustering jobs into small
    pieces, I suggest you just use "--L 1" unless you have
    enough RAM for each cpu
--S Number of segments to split input DB into, default 64
--Q number of jobs to submit to queue queuing system, default 0
    by default, the program use ssh mode to submit remote jobs
--T type of queuing system, "PBS", "SGE" are supported, default PBS
--R restart file, used after a crash of run
</code>

==== CD-HIT-2D-PARA ====
__** [CD-HIT-2D-PARA is no longer supported, since the multi-threaded cd-hit become available.] **__

CD-HIT-2D-PARA is a script that runs cd-hit-2d, cd-hit-est-2d in a parallel
mode. It splits the input databases; runs cd-hit-2d or cd-hit-est-2d in
parallel on a computer cluster; and finally merges the outputs into a single
file.  You can run it as you run cd-hit-2d or cd-hit-est-2d. The input is a
protein or DNA/RAN dataset in fasta format and the output are two files: a
fasta file of representative sequences and a text file of list of clusters. 

Basic command:
  cd-hit-para.pl -i nr -i2 swissprot -o swissprot_vs_nr -c 0.6 -n 4 --Q 20 -T "SGE" --S 2 --S2 20
  
where  
<code>
    --P  program, "cd-hit-2d" or "cd-hit-est-2d",
         default "cd-hit-2d"
    --B  filename of list of hosts,
         requred unless -Q or -L option is supplied
    --L  number of cpus on local computer, default 0
         when you are not running it over a cluster, you can use
         this option to divide a big clustering jobs into small
         pieces, I suggest you just use "--L 1" unless you have
         enough RAM for each cpu
    --S  Number of segments to split 1st db into, default 2
    --S2 Number of segments to split 2nd db into, default 8
    --Q  number of jobs to submit to queue queuing system, default 0
         by default, the program use ssh mode to submit remote jobs
    --T  type of queuing system, "PBS", "SGE" are supported, default PBS
    --R  restart file, used after a crash of run
     -h  print this help
</code>


==== Incremental clustering ====

It is easy to make incremental update with cd-hit /cd-hit-2d. For example:
  nr is the nr database of last month
  month is the new sequences of nr of this month

In last month, you ran:
  cd-hit -i nr -o nr90 -c 0.9 -n 5 -d 0 -M 16000 -T 16

This month, you can run incremental clustering
  cd-hit-2d -i nr90 -i2 month -o month-new -c 0.9 -n 5 -d 0 -M 16000 -T 16
  cd-hit -i month-new -o month90 -c 0.9 -n 5 -d 0 -M 16000 -T 16
  cat month90 >> nr90
  clstr_merge.pl nr90.clstr month-new.clstr > temp.clstr
  cat temp.clstr month90.clstr > this_month_nr90.clstr 

This approach is much faster than running from scratch. It also preserves
stable cluster structure. 

==== Hierarchically clustering ====

With multiple-step, iterated runs of CD-HIT, you perform a clustering in a
neighbor-joining method, which generates a hierarchical structure. The third step use psi-cd-hit, please see psi-cd-hit section for details. 

This way is faster than one-step clustering. It can also be more accurate. 

There is a problem with one-step clustering. Two very similar sequences A and B may be clustered into different clusters. For example, let the clustering threshold to be 60%, IAB (identity of AB) = 95%, IAC â¥ 60%, but IBC < 60%. If C was first selected a cluster representative, then A will be in cluster âCâ, but âBâ will not, resulting near identical AB to be in different clusters. Hierarchically clustering will reduce this problem.
    
{{ :cd-hit-figure4.png }}

Commands:
  cd-hit -i nr -o nr80 -c 0.8 -n 5 -d 0 -M 16000 -T 16
this generate nr80 and nr80.clstr

  cd-hit -i nr80 -o nr60 -c 0.6 -n 4 -d 0 -M 16000 -T 16
this use nr80 to generate nr60 and nr60.clstr
  
  psi-cd-hit.pl -i nr60 -o nr30 -c 0.3
this use nr60 to generate nr30 and nr30.clstr  
  
  clstr_rev.pl nr80.clstr nr60.clstr > nr80-60.clstr
nr60.clstr only lists sequences from nr80, script clstr_rev.pl add the original sequences from nr but not in nr80 into the output file nr80-60.clstr
  
  clstr_rev.pl nr80-60.clstr nr30.clstr > nr80-60-30.clstr
nr30.clstr only lists sequences from nr60, script clstr_rev.pl add the original sequences into file nr80-60-30.clstr



===== CD-HIT AuxTools =====

CD-HIT AuxTools is a set of auxiliary programs that can be used to assist the analysis
of the next generation sequencing data. It currently includes programs for removing
read duplicates, finding pairs of overlapping reads or joining pair-end reads etc.

==== cd-hit-dup ====


cd-hit-dup is a simple tool for removing duplicates from sequencing reads,
with optional step to detect and remove chimeric reads. When two files of paired end reads are used as inputs, each pair of reads will be concatenated into a single one. 
A number of options are provided to tune how the duplicates are removed.
Running the program without arguments should print out the list of available options,
as the following:

<code>
Usage:
cd-hit-dup -i input.fa -o output.fa [other options] (for single reads FASTQ)
cd-hit-dup -i input.fq -o output.fq [other options] (for single reads FASTA)
cd-hit-dup -i R1.fq -i2 R2.fq -o output-R1.fq -o2 output-R2.fq [other options] (for PE reads FASTQ)
cd-hit-dup -i R1.fa -i2 R2.fa -o output-R1.fa -o2 output-R2.fa [other options] (for PE reads FASTA)

Options:
    -i        Input file (FASTQ or FASTA);
    -i2       Second input file (FASTQ or FASTA);
    -o        Output file;
    -o2       Output file for R2;
    -d        Description length (default 0, truncate at the first whitespace character)
    -u        Length of prefix to be used in the analysis (default 0, for full/maximum length);
    -m        Match length (true/false, default true);
    -e        Maximum number/percent of mismatches allowed;
    -f        Filter out chimeric clusters (true/false, default false);
    -s        Minimum length of common sequence shared between a chimeric read
              and each of its parents (default 30, minimum 20);
    -a        Abundance cutoff (default 1 without chimeric filtering, 2 with chimeric filtering);
    -b        Abundance ratio between a parent read and a chimeric read (default 1);
    -p        Dissimilarity control for chimeric filtering (default 1);
</code>

=== Option details ===
<code>
    -u        Length of prefix to be used in the analysis (default 0, for full/maximum length);
</code>

For pair-end inputs, the program will take part (whole or prefix) of the first end 
and part (whole or prefix) of the second read,
and join them together to form a single read to do the analysis.
A positive value of this option specifies the length of the prefix to be taken from each read.
If a read is shorter than this length, letter 'N's will be appended to the read to make up
for the length.
When this option is not used or is used with a non-positive value,
the program will use the length of the longest read as the value of this option.

For single input analysis, only a positive value of this option will be effective.
It also allows the program to use only the prefix up to the specified length of each read
to do the analysis. In case that a read is shorter than this length, no 'N' is appended to
the read since it is not necessary.

<code>
    -m        Match length (true/false, default true);
</code>
"-m" specifies whether the lengths of two reads should be exactly the same to be considered
as duplicates.

<code>
    -e        Maximum number/percent of mismatches allowed;
</code>
Maximum number/percent of mismatches can be specified to control the similarity between two reads
for duplicate and chimeric detection. For duplicate detection, any two reads with number of mismatches
no greater than the specified value are considered to be duplicates. For chimeric detection,
this option control how similar a read should be to either of its parents.

<code>
    -f        Filter out chimeric clusters (true/false, default false);
</code>
This option specifies whether or not to carry out an additional step to filter out chimeric clusters.

<code>
    -s        Minimum length of common sequence shared between a chimeric read
              and each of its parents (default 30, minimum 20);
</code>
A read or cluster representative is considered as a potential chimeric only if it shares
at least the number of bases specified by this option with either of its parents.
This option is effective only if the option is set to true for filtering chimeric clusters.

<code>
    -a        Abundance cutoff (default 1 without chimeric filtering, 2 with chimeric filtering);
</code>
Each read is associated with an abundance number, which is the number of duplicates for the read.
cd-hit-dup always assumes the input contains duplicates and perform the duplicate detection step.
If no duplicate is found, the input is assumed to have duplicates remove in advance, and then,
the program will try to obtain the abundance information from the descriptions of the reads,
it interprets the number following "_abundance_" as the abundance number.

The abundance cutoff is mainly used for chimeric filtering to skip chimeric checking on reads with
abundance below this cutoff.

<code>
    -b        Abundance ratio between a parent read and a chimeric read (default 1);
</code>
This option specifies the abundance ratio between a parent read and a chimeric read. 
So for a read to be chimeric, either of its parents must have abundance at least as high as
the ratio times the abundance of the chimeric read.


<code>
    -p        Dissimilarity control for chimeric filtering (default 1);
</code>
Internally dissimilarity is measured by percent of mismatches with ungapped alignments.
By default the percentage cutoff is set to 0.01 (one percent).
This option specifies a multiplier to this percentage cutoff.
A higher value will increase the dissimilarity thresholds in chimeric filtering.


=== Output files ===

cd-hit-dup will output three or four files. Two of them are the same as the output files of CD-HIT:
one (named exactly the same as the file name specified by the "-o" option) is the cluster (or duplicate) representatives, the other is the clustering file (xxx.clstr) relating each duplicate
to its representative. For paired end reads, another file by the "-o2" option is the cluster representatives for R2 reads. 
The last file (xxx2.clstr) contains the chimeric clusters.
In this file, the description for each chimeric cluster contains cluster ids of its parent clusters
from the clustering file xxx.clstr.

=== Examples ===

== Duplicate Detection ==
Remove duplicates using default parameters:
<code>
cd-hit-dup -i input.fa -o output
</code>
By default, only reads that are identical are considered as duplicates.
If "-m" is set to false, duplicates will be allowed to have different length,
but the longer ones must have a prefix that is identical to the shorter ones.

<code>
cd-hit-dup -i input.fa -o output -u 50
</code>
This only compare the first 50 bases of all sequences, considering that in Illumina reads, sequence quality are better at the beginning of the reads.

Remove duplicates with a few mismatches:
<code>
cd-hit-dup -i input.fa -o output -e 2
cd-hit-dup -i input.fa -o output -e 0.01
</code>
The former will allow each duplicate read to have up to 2 mismatches when aligned to
its representative; and the later will allow up to one percent mismatches.

Remove duplicates from paired end reads:
<code>
cd-hit-dup -i pair-end1.fa -i2 pair-end2.fa -o output -o2 output-R2
</code>
Each read from "pair-end1.fa" and "pair-end2.fa" will be joint to form a single read to
detect duplicates. If they all are of the same length, the full length of each ends will
be used in forming the single read; otherwise, the default value of option "-u"
will be used to determine how the single read is created.

<code>
cd-hit-dup -i pair-end1.fa -i2 pair-end2.fa -o output -o2 output-R2 -u 50
</code>
This only consider the first 50 bases from both R1 and R2 reads.

Remove duplicates from paired end reads with control on how the paired end are jointed:
<code>
cd-hit-dup -i pair-end1.fa -i2 pair-end2.fa -o output -o2 output-R2 -u 100
</code>
With explicit "-u" options, any reads shorter than 100 will be padded with 'N's,
and the longer ones will be cut down to 100 base long. 
Then each pair of the 100 base long reads will be jointed to form a single 200 base long read.


== Chimeric Filtering ==

cd-hit-dup offers a very efficient way to detect chimeric reads. The basic idea is to find two parent
reads whose cross-over is sufficient similar to the chimeric read, while each single parent is 
sufficiently dissimilar to it. 

Such dissimilarity is measured by the percent of mismatches for no-gapped alignments.
For a given percentage "p" (from option "-p"), a chimeric read must share at least "p" 
percent mismatches with any other single read, namely, it much be sufficiently dissimilar to 
any single read.

For more robust detection of chimeric reads, a background percentage "p_bg" is calculated
as the mismatch percentage shared between the candidate chimeric read and the single read
that is most similar to the candidate. If "p_bg" is greater than "1.5*p", "1.5*p" will be
used as "p_bg" instead.

For a read to be classified as chimeric read, there must exist two reads/parents such that,
the leading part of the read is sufficiently similar to one parent, and the rest is sufficiently
similar to the other parent, with at most "p+p_bg" percent of mismatches in each part.
And the crossover between the two parents must share at most "p_bg" mismatches with the 
chimeric read.


Chimeric filtering with default parameters:
<code>
cd-hit-dup -i input.fa -o output -f true
</code>

Chimeric filtering with specified similarity level:
<code>
cd-hit-dup -i input.fa -o output -f true -p 1.5
</code>

Chimeric filtering with specified abundance difference:
<code>
cd-hit-dup -i input.fa -o output -f true -a 2
</code>
which means each parent of a chimeric read must be a least as twice abundant as the chimeric read.

Chimeric filtering will produce a cluster file named like "xxx2.clstr",
in which each cluster entry is a chimeric read/cluster. For example,
<code>
......
>Cluster 4 chimeric_parent1=2,chimeric_parent2=8
0   256nt, >FV9NWLF01CRIR3_abundance_23... *
>Cluster 5 chimeric_parent1=2,chimeric_parent2=0
0   250nt, >FV9NWLF01B4TBX_abundance_21... *
......
</code>
here "Cluster 5" contains a chimeric read "FV9NWLF01B4TBX", whose parents are identified
by cluster numbers "2" and "0" from the associated "xxx.clstr" file,
<code>
>Cluster 0
0   252nt, >FV9NWLF01ANLX2_abundance_2239... *
>Cluster 1
0   246nt, >FV9NWLF01C3KOB_abundance_1465... *
>Cluster 2
0   260nt, >FV9NWLF01AQOWA_abundance_1284... *
......
</code>
So the parent reads of the chimeric read "FV9NWLF01B4TBX" are "FV9NWLF01AQOWA" and "FV9NWLF01ANLX2".


==== cd-hit-lap ====

cd-hit-lap is program for extracting pairs of overlapping reads by clustering based on tail-head
overlaps (with perfect matching). The basic clustering strategy is the same as that in standard 
CD-HIT programs.
In this program, each read is clustered as either a "representative" or a "redundant" read.
For each "redundant" read, it must have a prefix that is identical a suffix of its representative read.

The options of this program can be obtained by running it it without any arguments:
<code>
[compute-0-0 cdhit-dup]$ ./cd-hit-lap
Options:
    -i        Input file;
    -o        Output file;
    -m        Minimum length of overlapping part (default 20);
    -p        Minimum percentage of overlapping part (default 0, any percentage);
    -d        Description length (default 0, truncate at the first whitespace character)
    -s        Random number seed for shuffling (default 0, no shuffling; shuffled before sorting by length);
    -stdout   Standard output type (default "log", other options "rep", "clstr");
</code>

The two options "-m" and "-p" can be used to control the minimum overlap that is required to
classify them as overlapping reads. Each pair of overlapping reads must have overlap length no less
than the threshold specified by "-m", and must also not be less than the length threshold computed
from the "-p" option.

Since the overlapping reads are searched using a greedy strategy, so different sortings of reads may
lead to different result. So it is advisable to run the program multiple times with read shuffling by different random number seeds, and then collect and merge the results.

Sometimes it may be more convenient to pipe the results of this program as stdout directly
to the stdin of other programs, to do this, the option "-stdout" can be used to choose
which type ("log" for program console information, "rep" for representative reads 
in FASTA or FASTQ format, "clstr" for the clustering output in CD-HIT format) of results 
to be writen to the stdout.

The output format of this program is the same as the standard CD-HIT. In the .clstr file, the alignment
positions indicate how the reads are overlapped. For example,
<code>
>Cluster 0
0   75nt, >1_lane2_624... *
1   75nt, >1_lane2_7169... at 1:65:11:75/+/100.00%
2   75nt, >1_lane2_36713... at 69:1:1:69/-/100.00%
3   75nt, >1_lane2_141482... at 1:56:20:75/+/100.00%
</code>
The cluster member #0 in cluster #0 is the representative of the cluster, and it overlaps with each
of the other members in the cluster. For cluster member #1, "1:65:11:75/+" tells that
the first 65 bases of member #1 overlaps with the last 65 bases of member #0;
"69:1:1:69/-" indicates that the last 69 bases of member #2 overlaps with 
the first 69 bases of member #0.


==== read-linker ====

read-linker is a very simple program to concatenate pair-end reads into single ones. It support the following options:
<code>
[compute-0-0 cdhit-dup]$ ./read-linker 
Options:
    -1 file       Input file, first end;
    -2 file       Input file, second end;
    -o file       Output file;
    -l number     Minimum overlapping length (default 10);
    -e number     Maximum number of errors (mismatches, default 1);
</code>
Only the pairs of reads that share at least a minimum overlapping length with mismatched no more
than the maximum number of errors, are jointed to form a single read.

===== PSI-CD-HIT clustering =====

The lowest threshold of CD-HIT is around 40%, in many applications, 
we need a much lower threshold, like 25%. Also CD-HIT-EST can not handle very long
sequences (e.g. genomes, scaffolds). 

PSI-CD-HIT clusters proteins at very low threshold, it also cluster long DNA 
sequences, through blastp, blastn and metablast. PSI-cd-hit is a Perl script, 
which runs similar incremental algorithm like CD-HIT, 
but using BLAST to calculate similarities. Below are the procedures of PSI-CD-HIT:

  - Sort sequences by decreasing length 
  - First one is the first representative
  - Using 1st one blast all remaining sequences, pick up its neighbors that meet the clustering threshold
  - Repeat until done

==== Installation ====
please download either legacy BLAST or BLAST+ and install the executables in your $PATH. The programs 
required by psi-cd-hit.pl are blastall, megablast, blastpgp and formatdb for legacy blast, and blastp, blastn, psiblast and makeblastdb for blast+.

==== Usage ====

Basic command:
  psi-cd-hit.pl -i nr60 -o nr30 -c 0.3
 
More options:
<code>
  input/output:
    -i  in_dbname, required
    -o  out_dbname, required
    -l  length_of_throw_away_sequences, default 10

  thresholds:
    -c  clustering threshold (sequence identity), default 0.3
    -ce clustering threshold (blast expect), default -1, 
        it means by default it doesn't use expect threshold, 
        but with positive value, the program cluster seqs if similarities
        meet either identity threshold or expect threshold 
    -G  (1/0) use global identity? default 1
        two sequences Long (i.e. representative) and Short (redunant) may have multiple
        alignment fragments (i.e. HSPs), see:
        seq1  xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx   Long sequence
                   ||||||||||||||||||             /////////////          i.e. representative
                   ||||||||||||||||||            /////////////                sequence
                   ||||||||HSP 1 ||||           ////HSP 2 ///
                   ||||||||||||||||||          /////////////
                   ||||||||||||||||||         /////////////
        seq2    xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx          Short sequence
                   <<  length 1    >>        <<   len 2 >>               i.e. redundant
                <<<<<<<<<<<< length of short sequence >>>>>>>>>>>>>>          sequence

                          total identical letters from all co-linear and non-overlapping HSPs
        Glogal identity = -------------------------------------------------------------------
                                        length of short sequence
        Local identity  = identity of the top high score HSP
        if you prefer to use -G 0, it is suggested that you also
        use -aS, -aL, such as -aS 0.8, to prevent very short matches.
    -aL	alignment coverage for the longer sequence, default 0.0
 	if set to 0.9, the alignment must covers 90% of the sequence
    -aS	alignment coverage for the shorter sequence, default 0.0
 	if set to 0.9, the alignment must covers 90% of the sequence
    -g  (1/0), default 0
        by cd-hit's default algorithm, a sequence is clustered to the first 
        cluster that meet the threshold (fast cluster). If set to 1, the program
        will cluster it into the most similar cluster that meet the threshold
        (accurate but slow mode)
        but either 1 or 0 won't change the representatives of final clusters
    -circle (1/0), default 0
        when set to 1, treat sequences as circular sequence.
        bacterial genomes, plasmids are circular, but their genome coordinate maybe arbitary, 
        the 2 HSPs below will be treated as non co-linear with -circle 0
        the 2 HSPs below will be treated as     co-linear with -circle 1
              -------------circle-----------    
              |                            |   
        seq1  xxxxxxxxxxxxxxxxxxxxxxxxxxxxxx      genome / plasmid 1 
               \\\\\\\\      /////////////         
                \\\\\\\\    /////////////                
                  HSP 2 -> ////HSP 1 ///   <-HSP 2
                          /////////////     \\\\\\\\
                         /////////////       \\\\\\\\
        seq2           xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx   genome / plasmid 2
                       |                             |
                       -----------circle--------------
   program:
     -prog (blastp, blastn, megablast, blastpgp), default blastp 
     -p  profile search para, default
           "-j 3 -F F -e 0.001 -b 500 -v 500"
     -dprof database for building PSSM, default using input
         you can also use another database that is more comprehensive like NR80
     -s  blast search para, default
           "-F F -e 0.000001 -b 100000 -v 100000"
     -bs (1/0) default 1
        pipe blast results from into parser instead of save in hard drive (save time)

   compute:
     -exec (qsub, local) default local
          this program writes a shell script to run blast, this script is
          either performed locally by sh or remotely by qsub
          with qsub, you can use PBS, SGE etc
     -host number of hosts for qsub 
     -core number of cpu cores per computer, default 1
     -shf a filename for add local settings into the job shell script
          for example, when you run PBS jobs, you can add quene name etc in this
          file and this script will add them into the job shell script
e.g. your file may have followings 
#PBS -v PATH
#PBS -l walltime=8:00:00
#PBS -q jobqueue

  job:
    -rs steps of save restart file and clustering output, default 5000
        everytime after process 5000 sequences, program write a 
        restart file and current clustering information
    -restart restart file, readin a restart file
        if program crash, stoped, termitated, you can restart it by
        add a option "-restart sth.restart"
    -rf steps of re format blast database, default 200,000
        if program clustered 200,000 seqs, it remove them from seq
        pool, and re format blast db to save time
    -J  job, job_file, exe specific jobs like parse blast outonly
        DON'T use it, it is only used by this program itself
    -k (1/0) keep blast raw output file, default 0

    -P path to executables

</code>

==== Examples ====

== Protein clustering ==
First, we use cd-hit to cluster the input down to 60% identity
<code>
cd-hit -i db    -o db_90 -c 0.9 -n 5 -g 1 -G 0 -aS 0.8  -d 0 -p 1 -T 16 -M 0 > db_90.log
cd-hit -i db_90 -o db_60 -c 0.6 -n 4 -g 1 -G 0 -aS 0.8  -d 0 -p 1 -T 16 -M 0 > db_60.log
</code>

Cluster on a single computer
<code>
  ./psi-cd-hit.pl -i db_60 -o db_30 -c 0.3 -ce 1e-6 -aS 0.8 -G 0 -g 1 -exec local -core 16
  clstr_rev.pl db_90.clstr db_60.clstr > db90-60.clstr
  clstr_rev.pl db90-60.clstr db_30.clstr > db90-60-30.clstr
</code>
Here, 
  -ce 1e-6 and -c 0.3 means cutoff at either 30% identity or 1e-6 e.value
  -G 0 means global identity
  -aS 0.8 means that alignment must cover 80% of shorter (redundant) sequence
  -g 1, slow but accurate mode, allowing sequences to be grouped to its most similar cluster
  -core 16, use 16 threads for blast search
  -clstr_rev.pl will combined all the cd-hit runs, see Hierarchically clustering

Cluster on a computer cluster (with qsub)
<code>
  ./psi-cd-hit.pl -i db_60 -o db_30 -c 0.3 -ce 1e-6 -aS 0.8 -G 0 -g 1 -exec qsub -host 8 -core 8 -shf qsub_sh_template
  clstr_rev.pl db_90.clstr db_60.clstr > db90-60.clstr
  clstr_rev.pl db90-60.clstr db_30.clstr > db90-60-30.clstr  
</code>

Restart:
<code>
  ./psi-cd-hit.pl -i db_60 -o db_30 -c 0.3 -ce 1e-6 -aS 0.8 -G 0 -g 1 -exec local -core 16 -restart db_30.restart 
  ./psi-cd-hit.pl -i db_60 -o db_30 -c 0.3 -ce 1e-6 -aS 0.8 -G 0 -g 1 -exec qsub -host 8 -core 8 -shf qsub_sh_template -restart db_30.restart
</code>
In case of program crash, it restart from where it stops without re-running blast searches.


== Clustering very long DNA sequences ==
<code>
  ./psi-cd-hit.pl -i db.fna -o db90.fna -c 0.9 -G 1 -g 1 -prog megablast -s "-F F -e 0.000001 -b 100000 -v 100000" -exec local -core 32
  ./psi-cd-hit.pl -i db.fna -o db90.fna -c 0.9 -G 1 -g 1 -prog blastn -circle 1 -exec local -core 32
</code>
Here,
  First example uses megablast
  Second example uses blastn
  -circle 1 means consider circular genome (see psi-cd-hit options above)
  

===== CD-HIT tools =====

==== cd-hit-div, cd-hit-div.pl ====

Both the executable binary program cd-hit-div and the perl script divide a FASTA file into pieces. The difference is that cd-hit-div sorts the sequences before dividing them while the perl script does not.

Commands:
  cd-hit-div -i input -o output -div n
  cd-hit-div.pl input output n

where "n" is the number of output files. The output files will be named as output-0, output-1 etc.
==== plot_len.pl ====

This is a script to print out distributions of clusters & sequences.  

Commands:
  plot_len.pl input.clstr \
  1,2-4,5-9,10-19,20-49,50-99,100-299,500-99999 \
  10-59,60-149,150-499,500-1999,2000-999999

where
  2nd line are sizes of cluster
  3rd line are lengths of sequences 

It will print distribution of clusters and sequences :

<code>
Size    # seq   #clstr  10-59   60-149  150-499 500-1999 2000-up
1       266312  266312  36066   103737  103285  22727   497
2-4     208667  81131   1229    14680   44607   20006   609
5-9     156558  24198   118     2148    12026   9388    518
10-19   155387  11681   30      596     5024    5462    569
20-49   176815  6007    6       139     2212    3135    515
50-99   106955  1568    0       24      410     955     179
100-499 154209  896     0       3       124     597     172
500-up  43193   40      0       0       1       14      25
Total   1268096 391833  37449   121327  167689  62284   3084
</code>

==== clstr_sort_by.pl ====

This script sort clusters in .clstr file by length, size

Commands:
  Clstr_sort_by.pl < input.clstr no > input_sort.clstr
Where, "no" means by size of the cluster


==== clstr_sort_prot_by.pl ====

This script sort sequences within clusters in .clstr file by length, name, etc.

Commands:
  Clstr_sort_prot_by.pl input.clstr id > input_sort.clstr
Where, "no" means by id of sequences


==== clstr_merge.pl ====

It merges two or more .clstr files. The cluster orders need to be identical.

Commands:
  cd-hit-2d -i db1 -i2 db2 -o db2new -c 0.9 -n 5
  cd-hit-2d -i db1 -i2 db3 -o db3new -c 0.9 -n 5
  clstr_merge.pl db2new.clstr db3new.clstr > db23new.clstr


==== clstr_merge_noorder.pl ====

It merges two or more .clstr files. The cluster orders do not have to be identical.

Commands:
  cd-hit-2d -i db1 -i2 db2 -o db2new -c 0.9 -n 5
  cd-hit-2d -i db1 -i2 db3 -o db3new -c 0.9 -n 5
  clstr_merge_noorder.pl db2new.clstr db3new.clstr > db23new.clstr

==== clstr_ renumber.pl ====

It renumbers clusters and sequences within clusters in .clstr file after merge
or other operations 

Commands:
  Clstr_renumber.pl input.clstr > input_ren.clstr

==== clstr_rev.pl ====

It combines a .clstr file with its parent .clstr file 

Commands:
  cd-hit -i nr -o nr90 -c 0.9 -n 5
  cd-hit -i nr90 -o nr60 -c 0.6 -n 4
  clstr_rev.pl nr90.clstr nr60.clstr > nr60_from90.clstr
  psi-cd-hit -i nr60 -o nr30 -c 0.3
  clstr_rev.pl nr60_from90.clstr nr30.clstr > nr30_from90.clstr 


==== make_multi_seq.pl ====

This script reads the .clstr file, it generates a separate fasta file for each cluster over certain size and saves it in designated subdirectory. To run this script correctly, "-d 0" option should be used in the cd-hit run and it is better to use "-g 1" in the cd-hit run to get accurate clustering results. For example,

Commands:
  cd-hit -i db -o dbout -c 0.6 -n 4 -d 0 -g 1
  make_multi_seq.pl seq_db dbout.clstr multi-seq 20

will generate fasta files in "multi-seq" directory for clusters with more than 20 member sequences. Files will be named as "clusterN" where "N" is serial number of a cluster.
==== clstr2xml.pl ====

This script converts a cluster file or combines multiple cluster files from a hierarchical cd-hit run to xml format. The output is sorted by sequence length (default) or cluster size. The input cluster files must be in the order of being generated, that is, the cluster file with higher identity cutoff comes first.

Command:
  clstr2xml.pl [-len|-size] input1.clstr [input2.clstr input3.clstr ...]
===== CD-HIT Web Server =====

The CD-HIT web server is available from [[http://cd-hit.org]]. All basic functions
of CD-HIT are provided through tab-based interfaces in our web server. For
CD-HIT and CD-HIT-EST, users can upload a FASTA file, select a desired
sequence identity level and other parameters. CD-HIT-2D (CD-HIT-EST-2D) can
compare two databases uploaded by users. H-CD-HIT and H-CD-HIT-EST in our
server performs hierarchical clustering up to 3 steps. 

The CD-HIT-454 web server is also available from [[http://cd-hit.org]]. 

===== Use cases =====
Here, a use case is defined as a sequence clustering related problem or application that cannot be easily solved with existing clustering approaches, such as CD-HIT. However, it is feasible to solve such a use case by customizing current clustering algorithms or utilizing current approach in a very intelligent way or non-standard manner. In the last years, we have developed many use cases in addressing various problems. We will release these use cases after additional testing. These use cases will be described in the following chapters.

===== CD-HIT-OTU-MiSeq =====
ï»¿This use case is developed for clustering 16S rRNA genes into OTUs for microbiome studies. In recent years, Illumina MiSeq sequencers became dominant in 16S rRNA sequencing. The Paired End (PE) reads need to be assembled first. However many reads can not be accurately assembled because the poor quality at the 3â ends of both PE reads in the overlapping region. This causes that many sequences are discarded in the analysis. CD-HIT-OTU-MiSeq has unique features to cluster MiSeq 16S sequences. 
  - The package can clustering PE reads without joining them into contigs. 
  - Users can choose a high quality portion of the PE reads for analysis (e.g. first 200 / 150 bases from forward / reverse reads), according to base quality profile. 
  - We implemented a tool that can splice out the target region (e.g. V3-V4) from a full-length 16S reference database into the PE sequences. CD-HIT-OTU-MiSeq can cluster the spliced PE reference database together with samples, so we can derive Operational Tax-onomic Units (OTUs) and annotate these OTUs concurrently. 
  - Chimeric sequences are effectively identified through both de novo and reference-based approaches. 

The most important unique feature of CD-HIT-OTU-MiSeq is to only use high quality region at the 5â ends of R1 and R2 reads. For example, the effective read length can be 200 bases for R1 and 150 bases for R2. The effective portions of PE reads are clustered together with spliced PE sequences from the reference database to derive OTUs (Figure).

{{:cd-hit-otu-miseq-figure-1.png|}}

==== Installation ====
First download and install full cd-hit package
  * download current CD-HIT at [[https://github.com/weizhongli/cdhit/releases]], for example cd-hit-v4.6.2-2015-0511.tar.gz
  * unpack the file with " tar xvf cd-hit-v4.6.2-2015-0511.tar.gz --gunzip"
  * change dir by "cd cd-hit-v4.6.2-2015-0511"
  * compile the programs by "make" with multi-threading (default), or by "make openmp=no" without multi-threading (on old systems without OpenMP)
  * cd cd-hit-auxtools
  * compile cd-hit-auxtools by "make"
  * CD-HIT-OTU-MiSeq scripts are inside a folder like cd-hit-v4.6.2-2015-0511/usecases/Miseq-16S

CD-HIT-OTU-MiSeq uses Trimmomatic for sequence quality control. It can be downloaded from [[http://www.usadellab.org/cms/?page=trimmomatic]] or [[https://github.com/timflutre/trimmomatic]]. We also have a copy at [[http://weizhongli-lab.org/download-data/cd-hit-otu-miseq/]].

  * modify NG-Omics-Miseq-16S.pl
Please edit usecases/Miseq-16S/NG-Omics-Miseq-16S.pl, in the top few lines:
  $CD_HIT_dir   = "PATH_to_cd-hit";
  $NGS_prog_trimmomatic = "PATH/trimmomatic-0.32.jar";  #### where you have installed Trimmomatic

==== Download reference and sample datasets ====
Reference database and sample datasets can be downloaded from [[http://weizhongli-lab.org/download-data/cd-hit-otu-miseq/]].

The reference database Greengene-13-5-99.fasta.gz was processed from original Greengene database, so that sequences with more specific annotations are at the beginning of the file. You need to download and gunzip it.

You can also download Greengene and generate it. You should download Greengene from [[http://greengenes.secondgenome.com/downloads]], or [[ftp://greengenes.microbio.me/]]. Please download file like greengenes_release/gg_13_5/gg_13_5_otus.tar.gz, unpack the tar file. You may find gg_13_5_otus/taxonomy/99_otu_taxonomy.txt and gg_13_5_otus/rep_set/99_otus.fasta. There is a script: usecases/Miseq-16S/greengene-ann1.pl.  

Commands:
  /greengene-ann1.pl -i gg_13_5_otus/taxonomy/99_otu_taxonomy.txt -j gg_13_5_otus/rep_set/99_otus.fasta -o Greengene-13-5-99.fasta

The Miseq-otu-example.tar.gz contains two Miseq 16S samples. You can download and unpack to test.

==== Usage ====

**Step 1. prepare fastq files and sample file:** Most projects have multiple samples sequenced at the same region. You should already have paired ended fastq files for these samples, put them in a working directory in similar way as the testing datasets, where the R1.fq and R2.fq are placed in separate folder for each sample. So in the working directory, you should have files:
  sample_name_1/R1.fq
  sample_name_1/R2.fq
  sample_name_2/R1.fq
  sample_name_2/R2.fq
  ...
  sample_name_N/R1.fq
  sample_name_N/R2.fq
  
Then, please prepare a sample file in the working directory. The file should look like:
  sample_name_1 R1.fq R2.fq
  sample_name_2 R1.fq R2.fq
  sample_name_N R1.fq R2.fq

**Step 2. Reference database preparation:** We implemented a tool that can splice out the target amplicon region (e.g. V3-V4) from a full-length 16S rRNA reference sequence database, such as Greengene, RDP and Silva, into PE sequences. If there are multiple samples in a project sequenced with the same amplicon of same variable region, only one spliced reference database is needed. To run:

  path_to_cd-hit_dir/usecases/Miseq-16S/16S-ref-db-PE-splice.pl -i sample_name_1/R1.fq -j sample_name_2/R2.fq  -d Greengene-13-5-99.fasta -o gg_13_5-PE99.150-100 -p 150 -q 100 -c 0.99
Where Greengene-13-5-99.fasta is our re-formatted Greengene sequence file. This program will output spliced PE files gg_13_5-PE99.150-100-R1 and gg_13_5-PE99.150-100-R2.

**Step 3. Run sequence QC and OTU clustering for each sample:**. In the working directory, run
  PATH_to_cd-hit-dir/usecases/NG-Omics-WF.pl -i PATH_to_cd-hit-dir/usecases/NG-Omics-Miseq-16S.pl -s sample_file -T otu:150:100:0.97:0.0001:PATH_to-gg_13_5-PE99.150-100-R1:PATH_to-gg_13_5-PE99.150-100-R2:75 -J write-sh
where: 150 and 100 are the effective length, 0.97 is the OTU clustering cutoff, 0.00001 is the abundance cutoff, 75 is the length for chimeric checking at each R1 and R2 read

This command will generate shell scripts for QC and for OTU for each sample. The scripts will be in WF-sh folder. You can first run the qc.sample_name.sh and then run otu.sample_name.sh 
  
NG-Omics-WF.pl [[https://github.com/weizhongli/ngomicswf]] is a very powerful workflow and pipeline tool developed in our group. It is not fully released yet, since we need more time to document this tool. However, you can try to use NG-Omics-WF.pl to automatically run all your samples. First edit NG-Omics-Miseq-16S.pl and modify cores_per_node around line #36, then
  nohup PATH_to_cd-hit-dir/usecases/NG-Omics-WF.pl -i PATH_to_cd-hit-dir/usecases/NG-Omics-Miseq-16S.pl -s sample_file -T otu:150:100:0.97:0.0001:PATH_to-gg_13_5-PE99.150-100-R1:PATH_to-gg_13_5-PE99.150-100-R2:75 &
  
After the job finished, the OTU results will be in sample_name/otu folder, important files include
  * OTU.clstr: file lists all clusters and sequences 
  * removed_chimeric*: chimeric sequenced removed 
  * small_clusters.list: low abundance small clusters removed

**Step 4. pool all the samples together:** Please run
  PATH_to_cd-hit-dir/usecases/pool_samples.pl -s sample_file -o pooled_sample. 
This will pool sequences from all sample and re-run OTU clustering. We can pool hundred of samples without problem. After job finished, additional files will be available from pooled_sample directory
  * OTU.clstr: file list all clusters and sequences from all samples
  * removed_chimeric*: chimeric sequenced removed 
  * small_clusters.list: low abundance small clusters removed
  * OTU.txt: spread sheet list number of sequences in each OTU for each sample, it also show annotation for each OTU.
  * OTU.biome: OTU.txt in biome format
  
  

===== References =====

If you find cd-hit helpful to your research and study, please kindly cite the
relevant references from the list below. 
  - Weizhong Li, Lukasz Jaroszewski & Adam Godzik. Clustering of highly homologous sequences to reduce the size of large protein databases. Bioinformatics (2001) 17:282-283, [[http://bioinformatics.oupjournals.org/cgi/reprint/17/3/282.pdf|PDF]], [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11294794|Pubmed]]
  - Weizhong Li, Lukasz Jaroszewski & Adam Godzik. Tolerating some redundancy significantly speeds up clustering of large protein databases. Bioinformatics (2002) 18: 77-82, [[http://bioinformatics.oupjournals.org/cgi/reprint/18/1/77.pdf|PDF]], [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11836214|Pubmed]]
  - Weizhong Li & Adam Godzik. Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics (2006) 22:1658-1659, [[http://bioinformatics.oxfordjournals.org/cgi/reprint/22/13/1658|PDF]], [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=Abstract&list_uids=16731699|Pubmed]]
  - Ying Huang, Beifang Niu, Ying Gao, Limin Fu and Weizhong Li. CD-HIT Suite: a web server for clustering and comparing biological sequences. Bioinformatics, (2010). 26:680 [[http://bioinformatics.oxfordjournals.org/cgi/reprint/btq003v1|PDF]] [[http://www.ncbi.nlm.nih.gov/pubmed/20053844|Pubmed]]
  - Beifang Niu, Limin Fu, Shulei Sun and Weizhong Li, Artificial and natural duplicates in pyrosequencing reads of metagenomic data. BMC Bioinformatics, (2010), 11:187 [[http://www.biomedcentral.com/content/pdf/1471-2105-11-187.pdf|PDF]] [[http://www.ncbi.nlm.nih.gov/pubmed/20388221|Pubmed]]
  - Limin Fu, Beifang Niu, Zhengwei Zhu, Sitao Wu and Weizhong Li, CD-HIT: accelerated for clustering the next generation sequencing data. Bioinformatics, (2012), 28 (23): 3150-3152. doi: 10.1093/bioinformatics/bts565, [[http://bioinformatics.oxfordjournals.org/content/28/23/3150|PDF]], [[http://www.ncbi.nlm.nih.gov/pubmed/23060610|Pubmed]]
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