The make.contigs command reads a forward fastq file and a reverse fastq file and outputs new fasta and report files.
The make.contigs command parameters are file, ffastq, rfastq, ffasta, rfasta, fqfile, rqfile, findex, rindex, oligos, format, tdiffs, bdiffs, pdiffs, align, match, mismatch, gapopen, gapextend, insert, deltaq, maxee, allfiles and processors.
mothur > make.contigs(ffastq=test_1.fastq, rfastq=test_2.fastq)
mothur > make.contigs(ffastq=test_1.fastq.gz, rfastq=test_2.fastq.gz)
mothur > make.contigs(file=combo.file)
mothur > make.contigs(ffastq=forward.fastq, rfastq=reverse.fastq, findex=forwardIndex.fastq, rindex=reverseIndex.fastq)
mothur > make.contigs(ffasta=test_1.fasta, rfasta=test_2.fasta, rqfile=test_1.qual, fqfile=test_1.qual)
This command will create a test_1.contigs.trim.fasta, test_1.contigs.scrap.fasta, test_1.trim.contigs.report and test_1.scrap.contigs.report file.
The file parameter is 2, 3 or 4 column file containing the fastq files. Tired of creating the file file? Let mothur help with the make.file command!
The 2 column format consists of the forward fastq file in the first column and their matching reverse fastq files in the second column. This type can be used with an oligos file create a group file for your dataset.
small.forward.fastq small.reverse.fastq test.forward2.fastq test.reverse2.fastq ...
The 3 column format is used for datasets where the sequences have already had the barcodes and primers removed and been split into separate files. The first column is the group, the second is the forward fastq and the third column contains the reverse fastq.
F8D0 F8D0_S345_L001_R1_001.fastq F8D0_S345_L001_R2_001.fastq F8D125 F8D125_S358_L001_R1_001.fastq F8D125_S358_L001_R2_001.fastq F8D141 F8D141_S359_L001_R1_001.fastq F8D141_S359_L001_R2_001.fastq F8D142 F8D142_S360_L001_R1_001.fastq F8D142_S360_L001_R2_001.fastq F8D143 F8D143_S361_L001_R1_001.fastq F8D143_S361_L001_R2_001.fastq F8D144 F8D144_S362_L001_R1_001.fastq F8D144_S362_L001_R2_001.fastq F8D145 F8D145_S363_L001_R1_001.fastq F8D145_S363_L001_R2_001.fastq F8D146 F8D146_S364_L001_R1_001.fastq F8D146_S364_L001_R2_001.fastq F8D147 F8D147_S365_L001_R1_001.fastq F8D147_S365_L001_R2_001.fastq F8D148 F8D148_S366_L001_R1_001.fastq F8D148_S366_L001_R2_001.fastq F8D149 F8D149_S367_L001_R1_001.fastq F8D149_S367_L001_R2_001.fastq F8D150 F8D150_S368_L001_R1_001.fastq F8D150_S368_L001_R2_001.fastq ...
The 4 column format is for use with index files. The format is forward fastq file then reverse fastq then forward index and reverse index file. If you only have one index file add ‘none’ for the other one. Here’s an example with just a reverse index file:
My.forward.fastq My.reverse.fastq none My.index.fastq
mothur will process each pair and create a combined fasta and report file with all the sequences.
ffastq & rfastq
The ffastq and rfastq parameters are used to provide a forward fastq and reverse fastq file to process. If you provide one, you must provide the other.
ffasta & rfasta
The ffasta and rfasta parameters are used to provide a forward fasta and reverse fasta file to process. If you provide one, you must provide the other.
fqfile & rqfile
The fqfile and rqfile parameters are used to provide a forward quality and reverse quality files to process with the ffasta and rfasta parameters. If you provide one, you must provide the other.
findex & rindex
The findex and rindex parameters are used to provide a forward index and reverse index files to process. If you use an index file, you must provide an oligos file.
The index file is a fastq file containing barcodes for the reads. It looks like:
@M00704:50:000000000-A3G0K:1:1101:15777:1541 1:N:0:0 NAGAGAGGATCT + #>>3A3A>CFFF @M00704:50:000000000-A3G0K:1:1101:15370:1541 1:N:0:0 NAGAGAGGATCT + #>>ABCCCFFFF ...
The oligos option takes a file that can contain the sequences of the paired primers and barcodes and their sample identifier. Each line of the oligos file can start with the key words “primer” and “barcode” or it can start with a “#” to tell mothur to ignore that line of the oligos file. Here’s an example:
primer CCTACGGGAGGCAGCAG ATTACCGCGGCTGCTGG V3 primer ATTAGAWACCCBDGTAGTCC CCCGTCAATTCMTTTRAGT V5 primer ACTYAAAKGAATTGACGGG ACRACACGAGCTGACGAC V6 BARCODE ccaac cactg F01R2A BARCODE ccaac aacca F01R2B BARCODE ccaac tgtca F01R2C BARCODE ccaac aaacc F01R2D ... mothur > make.contigs(ffastq=test_1.fastq, rfastq=test_2.fastq, oligos=test.oligos)
bdiffs & pdiffs & tdiffs
These parameters are used to allow differences in the barcode and primers. pdiffs is maximum number of differences to the primer sequence, default=0. bdiffs is maximum number of differences to the barcode sequence, default=0. tdiffs is maximum total number of differences to the barcode and primer (default to pdiffs + bdiffs). To clarify, the make.contigs command with pdiffs=2 sets the total allowed primer diffs to 2. mothur will allow for a max of 2 diffs on the forward read and 2 diffs on the reverse read, but the total number of primer diffs cannot exceed 2. This means if you have a read with 2 diffs on the forward and 2 diffs on the reverse, the total primer diffs are 4, and the read would be scrapped.
If you are running the make.contigs command with paired barcodes or primers, you can use the checkorient parameter. When checkorient=t and mothur can’t find the barcodes and primers, it will search the reverse compliment. The default is true. Let’s look at a small example:
The oligos file looks like:
primer CCTACGGGAGGCAGCAG ATTACCGCGGCTGCTGG V3 primer ATTAGAWACCCBDGTAGTCC CCCGTCAATTCMTTTRAGT V5 primer ACTYAAAKGAATTGACGGG ACRACACGAGCTGACGAC V6 BARCODE ccaac cactg F01R2A BARCODE ccaac aacca F01R2B BARCODE ccaac tgtca F01R2C BARCODE ccaac aaacc F01R2D ...
When checkorient=t, mothur will also search for these primer and barcode combintaions:
paired reoriented primers - V3 ATTACCGCGGCTGCTGG CCTACGGGAGGCAGCAG V5 CCCGTCAATTCMTTTRAGT ATTAGAWACCCBDGTAGTCC V6 ACRACACGAGCTGACGAC ACTYAAAKGAATTGACGGG paired reversed primers - V3 CTGCTGCCTCCCGTAGG CCAGCAGCCGCGGTAAT V5 GGACTACHVGGGTWTCTAAT ACTYAAAKGAATTGACGGG V6 CCCGTCAATTCMTTTRAGT GTCGTCAGCTCGTGTYGT paired reoriented barcodes - F01R2A CACTG CCAAC F01R2B AACCA CCAAC F01R2C TGTCA CCAAC F01R2D AAACC CCAAC paired reversed barcodes - F01R2A GTTGG CAGTG F01R2B GTTGG TGGTT F01R2C GTTGG TGACA F01R2D GTTGG GGTTT
NOTE: If the reversed or reoriented primers / barcodes are found, mothur assumes the sequence fragments are reversed and flips them before assembly.
oligos scrap code meanings
When you run mothur with an oligos file and the barcodes and or primers are not found mothur will assign the read to the *scrap file. mothur includes scrap codes to indicate why the read failed. Let’s look at an example:
- seqName | failureCode(checkorientFailureCode) ee=expectedError fbdiffs=forwardBarcodeDiffs(forwardBarcodeResult), rbdiffs=reverseBarcodeDiffs(reverseBarcodeResult), fpdiffs=forwardPrimerDiffs(forwardPrimerResult), rpdiffs=reversePrimerDiffs(reversePrimerResult)
- failureCode and checkOrientFailureCode: b(barcode), f(primer), t(totalDiffs)
- forwardBarcodeResult, reverseBarcodeResult, forwardPrimerResult,
reversePrimerResult options: match, multipleMatches, noMatch,
- match - mothur found the barcode or primer. Alternatively, no barcode or primer.
- multipleMatches - more than one barcode or primer matches this read.
- noMatch - mothur did not find a barcode or primer that matches this read.
- shortSeq - the read is shorter than the barcode and primer, no match.
M00278_49_000000000-CPBKN_1_1108_23033_3567 | b(b) ee=6.87543 fbdiffs=3(noMatch), rbdiffs=1001(noMatch) fpdiffs=0(match), rpdiffs=0(match)
fbdiffs=3(noMatch) - mothur found a barcode, but the number of diffs=3, bdiffs=1 so no match is found. rbdiffs=1001(noMatch) - 1001 indicates the reverse barcode was not searched for because the forward barcode was not found. (1000+bdiffs) = 1001 - no search and noMatch. fpdiffs=0(match) - perfect match to forward primer or no primers in oligos file. rpdiffs=0(match) - perfect match to reverse primer or no primers in oligos file.
maxambig && maxhomop && maxlength
The maxlength parameter allows you to screen reads based on length. The maxambig parameter allows you to screen reads with too many ambiguous bases. The maxhomop parameter allows you to screen reads with excessively long homopolymers.
To remove sequences with ambiguous bases or sequences longer than 275 bases run the following:
mothur > make.contigs(file=combo.file, maxambig=0, maxlength=275)
The format parameter is used to indicate whether your sequences are sanger, solexa, illumina1.8+ or illumina, default=illumina1.8+.
The align parameter allows you to specify the alignment method to use. Your options are: gotoh and needleman. The default is needleman.
mothur > make.contigs(ffastq=test_1.fastq, rfastq=test_2.fastq, align=gotoh)
match & mismatch & gapopen & gapextend
These parameters are used while aligning the sequence read to determine the overlap. The match parameter allows you to specify the bonus for having the same base. The default is 1.0. The mistmatch parameter allows you to specify the penalty for having different bases. The default is -1.0. The gapopen parameter allows you to specify the penalty for opening a gap in an alignment. The default is -2.0. The gapextend parameter allows you to specify the penalty for extending a gap in an alignment. The default is -1.0.
The insert parameter allows you to set a quality scores threshold. When we are merging the overlapping regions, in the case where we are trying to decide whether to keep a base or remove it because the base is compared to a gap in the other fragment, if the base has a quality score below the threshold we eliminate it. Default=20.
The deltaq parameter allows you to specify the delta allowed between quality scores of a mismatched base. For example in the overlap, if deltaq=5 and in the alignment seqA, pos 200 has a quality score of 30 and the same position in seqB has a quality score of 20, you take the base from seqA (30-20 >= 5). If the quality score in seqB is 28 then the base in the consensus will be an N (30-28<5) The default is 6.
The maxee parameter allows you to specify the maximum number of errors to allow in a sequence. Makes sense to use with deltaq=0. The expected number of errors is based on Edgar’s approach used in USEARCH/VSEARCH.
With the barcodes it is possible to sequence many different samples in a single run. It may happen that some of these samples should not be analyzed together for your analysis. To parse apart the sequences that belong to each sample, use the allfiles option. This will generate a fasta and groups file for each barcode defined in your oligos file:
The qfile parameter is used to indicate you want a quality file assembled. Default=false. NOTE: The assembled quality scores outputted by mothur cannot be used for downstream quality screening. The score calculations are modeled after pandseq’s method. Here’s a link to the explanation from their documentation, https://github.com/neufeld/pandaseq#the-scores-of-the-output-bases-seem-really-low-whats-wrong.
The trimoverlap parameter allows you to trim the sequences to only the overlapping section. The default is F. Use this option if you have 2x300bp sequences that are longer than your amplicon.
Renames sequences to reduce file sizes and greatly reduce the size of the column formatted distance matrix downstream. Uses the rename.seqs command to rename which creates a map file so you can revert to original names at any time. Default=F.
The processors parameter allows you to specify how many processors you would like to use. Default processors=Autodetect number of available processors and use all available.
Assembled Quality Scores
The assembled quality scores outputted by mothur cannot be used for downstream quality screening. The score calculations are modeled after pandseq’s method. Here’s a link to the explanation from their documentation, https://github.com/neufeld/pandaseq#the-scores-of-the-output-bases-seem-really-low-whats-wrong.
- 1.26.0 - First Introduced
- 1.30.0 - Officially released
- 1.31.2 - Bug Fix: - https://forum.mothur.org/viewtopic.php?f=3&t=2451
- 1.32.0 - Bug Fix: if file option is used with group provided, and one or more files contain less good reads than number of processors, group assignments were incorrect. - https://forum.mothur.org/viewtopic.php?f=4&t=2571&p=7025#p7025
- 1.32.0 - Added findex and rindex parameters. Modified file options to add 4 column option - ffastq rfastq findex rindex. May use NONE as option for findex and rindex. Modified oligos file option to allow none as option for paired barcodes for index files. BARCODE NONE GCTGATGAGCTG Group1 would indicate you have a reverse index file, but no forward index file.
- 1.34.0 - Added checkorient parameter. - https://forum.mothur.org/viewtopic.php?f=3&t=2993.
- 1.36.0 - Bug Fix: when using index files in version 1.35 quality data was over trimmed by the length of the barcode.
- 1.36.0 - allow for missing reads in files.
- 1.36.0 - allow for gzipped version of fastq files as inputs. Mac and Linux only.
- 1.37.0 - Adds rename parameter. #132
- 1.37.0 - Bug Fix: File mismatch error with certain sequence name formats and multiple processors. Three column format gz file not producing group file.
- 1.37.0 - Bug Fix: Fixes name mismatch error for rare names and gives a slight speed boost. #167
- 1.38.0 - Removes indexFile requirement for using NONE option in oligos file.
- 1.38.0 - Skips blanks files and continues.
- 1.38.1 - Fixes filename expansion issue. https://forum.mothur.org/viewtopic.php?f=3&t=5869&p=14857#p14857
- 1.39.0 - Fixes bug where mothur was not finding matches for sequence names “off by one character” in the name.
- 1.40.0 - Rewrite of threaded code. Default processors=Autodetect number of available processors and use all available.
- 1.40.2 - Fixes *contigs.report file append issue that resulted in reads missing from report.
- 1.40.5 - Fixes gz file read issues. #471
- 1.40.5 - Change to quality score calculation. #468
- 1.41.0 - Updates boost version to 1.68.0.
- 1.41.0 - Fixes group file issue with *.gz files. #480
- 1.41.0 - Fixes group names appending issue. https://forum.mothur.org/t/make-contigs-with-oligo-creating-new-names/3542
- 1.43.0 - Adds qfile option to make.contigs. #650
- 1.43.0 - Adds auto decompress feature to make.contigs if gz read fails. #634
- 1.43.0 - Windows users can now run make.contigs with *.gz files.
- 1.44.0 - Improves mothur’s checkorient option in make.contigs. Also sets checkorient=t by default.
- 1.45.0 Fixes bug with qfile option in make.contigs. #745
- 1.45.0 Fixes bug with make.contigs not making group file for *.gz files with oligos file. #753
- 1.45.0 Fixes make.contigs segfault.
- 1.47.0 Changes output file from group to count. #793
- 1.47.0 Adds maxambig, maxhomop, maxlength parameters to make.contigs. #793
- 1.48.0 Fixes bug with make.contigs screening parameters if maxlength not set