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The File file 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.

Note: You can add comments to the file file, or ignore files by adding a '#' to the beginning of the line.

#My sample comment ...
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 - ignore F8D125 sample
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