The make.sra command creates the necessary files for a NCBI submission. The xml file and individual sff or fastq files parsed from the original sff or fastq file. See Creating a new submission to see how to use make.sra in your workflow.

Default settings

The sra command can be run in 3 ways: with a sff file, fastq file or file option. The project and mimarksfile are also required. When you run the sra command with an sff file the oligos parameter is required.

mothur > get.mimarkspackage(oligos=GHL4YHV01.oligos)
mothur > make.sra(sff=GHL4YHV01.sff, oligos=GHL4YHV01.oligos, project=test.project, mimark=MIMarksData.txt)

or you can run the command with your fastq file. The oligos parameter is also required for this option.

mothur > get.mimarkspackage(oligos=test.oligos)
mothur > make.sra(fastq=test.fastq, oligos=test.oligos, project=test.project, mimark=MIMarksData.txt)

or you can run the sra command with the file option.

mothur > get.mimarkspackage(file=test.file)
mothur > make.sra(file=test.file, project=test.project, mimark=MIMarksData.txt)



The file parameter is used to provide a file containing a list of individual fastq or sff files or paired fastq files with a group assignment. File lines can be 2 or 3 columns. The 2 column files are sff file then oligos or fastqfile then oligos or ffastq and rfastq. You may have multiple lines in the file. The 3 column files are for paired read libraries. The format is groupName, forwardFastqFile reverseFastqFile. If you are running the command with a 2 column file and ffastq and rfastq you must provide an oligos file or mothur will assume your rfastq file is an oligos file.

Here’s an example of the 2 column form with sff files:

G3BMWHG01.sff   G3BMWHG01.oligos
GHL4YHV01.sff     GHL4YHV01.oligos
GHMDAJD01.sff    GHMDAJD01.oligos
GO5715J01.sff     GO5715J01.oligos
GQY1XT001.sff    GQY1XT001.oligos
GZGO5KL01.sff    GZGO5KL01.oligos

Here’s an example of the 2 column form with a fastq file:

test.ccs.fastq   barcodes.oligos

Here’s an example of the 2 column form with a paired fastq files. Note: This option must be run with an oligos file or mothur will assume the reverse.fastq file is an oligos file:

forward1.fastq   reverse1.fastq
forward2.fastq   reverse2.fastq

Here is an example of the 3 column form with fastq files:

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

or for single fastq files assigned to a group

F8D0   F8D0.fastq  NONE
F8D125 F8D125.fastq    NONE
F8D141 F8D141.fastq    NONE

Here’s an example of a 4 column file using index files:

My.forward.fastq My.reverse.fastq NONE My.rindex.fastq 

NONE is an option is no forward or reverse index file.


The project parameter allow you to provide your The project_file . It is required.


The mimark parameter is required. You can generate a mimark template for your groups using the get.mimarkspackage command. Please note, NCBI has controlled language to ensure uniform submissions. Unknown or inapplicable fields MUST be assigned ‘missing’ value.

mothur > get.mimarkspackage(oligos=GHL4YHV01.oligos)
mothur > make.sra(sff=sff=GHL4YHV01.sff, oligos=GHL4YHV01.oligos, project=test.project, mimark=MIMarksData.txt)


The oligos option takes a file that can contain the sequences of the forward and reverse primers and barcodes and their sample identifier. Each line of the oligos file can start with the key words “forward”, “reverse”, and “barcode” or it can start with a “#” to tell mothur to ignore that line of the oligos file. Here’s a link to more information about mothur’s[ [oligos_file]]

mothur > make.sra(sff=GHL4YHV01.sff, GHL4YHV01.oligos)

bdiffs & pdiffs & ldiffs & sdiffs & tdiffs

These parameters are used to allow differences in the barcode, primers, linkers and spacers. pdiffs is maximum number of differences to the primer sequence, default=0. bdiffs is maximum number of differences to the barcode sequence, default=0. ldiffs is maximum number of differences to the linker sequence, default=0. sdiffs is maximum number of differences to the spacer sequence, default=0. tdiffs is maximum total number of differences to the barcode, primer, linker and spacer (default to pdiffs + bdiffs + ldiffs + sdiffs).

mothur > make.sra(sff=GHL4YHV01.sff, GHL4YHV01.oligos, pdiffs=2, bdiffs=1)


The platform parameter is used to specify platform you are using choices are: _LS454,ILLUMINA,ION_TORRENT,PACBIO_SMRT. Default=_LS454. This is a controlled vocabulary section in the XML file that will be generated.


The orientation parameter is used to specify sequence orientation. Choices are: forward and reverse. Default=forward. This is a controlled vocabulary section in the XML file that will be generated.


The libstrategy parameter is used to specify library strategy. Default=AMPLICON. Choices are AMPLICON,WGA,WGS,WGX,RNA-Seq,miRNA-Seq,WCS,CLONE,POOLCLONE,CLONEEND,FINISHING,ChIP-Seq,MNase-Seq,DNase-Hypersensitivity,Bisulfite-Seq,Tn-Seq,EST,FL-cDNA,CTS,MRE-Seq,MeDIP-Seq,MBD-Seq,OTHER. This is a controlled vocabulary section in the XML file that will be generated.




The libsource parameter is used to specify library source. Default=METAGENOMIC. Choices are METAGENOMIC,GENOMIC,TRANSCRIPTOMIC,METATRANSCRIPTOMIC,SYNTHETIC,VIRAL_RNA,OTHER. This is a controlled vocabulary section in the XML file that will be generated.


The libselection parameter is used to specify library selection. Default=PCR. Choices are PCR,RANDOM,RANDOM_PCR,RT-PCR,HMPR,MF,CF-S,CF-H,CF-T,CF-M,MDA,MSLL,cDNA,ChIP,MNase,DNAse,Hybrid_Selection,Reduced_Representation,Restriction_Digest,5-methylcytidine_antibody,MBD2_protein_methyl-CpG_binding_domain,CAGE,RACE,size_fractionation,Padlock_probes_capture_method,other,unspecified. This is a controlled vocabulary section in the XML file that will be generated.


The instrument parameter is used to specify instrument. Choices are 454_GS-454_GS_20-454_GS_FLX-454_GS_FLX_Titanium-454_GS_Junior-Illumina_Genome_Analyzer-Illumina_Genome_Analyzer_II-Illumina_Genome_Analyzer_IIx-Illumina_HiSeq_2000-Illumina_HiSeq_1000-Illumina_MiSeq-PacBio_RS-Ion_Torrent_PGM-unspecified. Default=454_GS. This is a controlled vocabulary section in the XML file that will be generated.


The includescrap parameter is used to indicate whether or not to include the scrapped sequences in your submission. The default is true.


The trim parameter allows you to indicate if you would like a sequences and quality scores trimmed to the clipQualLeft and clipQualRight values when using sff files. Default=True.


  • 1.34.0 First Introduced
  • 1.36.0 Allow for assigning multiple sets of files to the same group in 3 column format.
  • 1.37.0 Bug Fix: error about no inputs selected when running make.sra with single fastq file and oligos file.
  • 1.37.0 Adds includescrap parameter
  • 1.39.0 Adds trim parameter for use with sff files
  • 1.40.0 Adds additional format checks. #410
  • 1.45.0 Fixes bug with sffinfo parsing with oligos option. #726 #727 Effects make.sra command as well because make.sra calls with an oligos file.

  • 1.48.0 Fixes bug with make.sra single-end reads