oligos file

The oligos file is used to provide barcodes and primers to mothur. mothur removes items in the following order: linkers, barcodes, spacers then primers.

Forward Primers

The forward primer lines can have two formats.

forward primerString optionalPrimerName

example

forward CCGTCAATTCMTTTRAGT

forward CCGTCAATTCMTTTRAGT  Group1

Reverse Primers

Reverse primers have only one format.

reverse reversePrimerString

example

reverse CCGTCAATTCMTTTRAGT

Paired Primers

The paired primer lines can have two formats.

primer forwardPrimerString reversePrimerString optionalPrimerName

example

primer ATTAGAWACCCBDGTAGTCC  CCCGTCAATTCMTTTRAGT

primer ATTAGAWACCCBDGTAGTCC  CCCGTCAATTCMTTTRAGT  V5

or to indicate forward only when paired reads

primer ATTAGAWACCCBDGTAGTCC  NONE  V5

or to indicate reverse only when paired reads

primer NONE  CCCGTCAATTCMTTTRAGT  V5

Barcodes

Barcodes should be formatted as follows:

barcode barcodeString sampleName

example

barcode    AATGGTAC    F003D000
barcode    AACCTGGC    F003D002
barcode    TTCGTGGC    F003D004
barcode    TTCTTGAC    F003D006
barcode    TTCGCGAC    F003D008
barcode    TCCAGAAC    F003D142
barcode    AAGGCCTC    F003D144
barcode    TGACCGTC    F003D146
barcode    AGGTTGTC    F003D148
barcode    TGGTGAAC    F003D150
barcode    AACCGTGTC   MOCK.GQY1XT001

Paired Barcodes

Paired Barcodes should be formatted as follows:

barcode forwardBarcodeString reverseBarcodeString sampleName

example

barcode  ccaac  ccaac  V1F01V9RA
barcode  ggttg  ccaac  V1F02V9RA
barcode  ttggt  ccaac  V1F03V9RA
...

or to indicate forward only when paired reads

barcode  ccaac  NONE  V1F01V9RA
barcode  ggttg  NONE  V1F02V9RA
barcode  ttggt  NONE  V1F03V9RA
...

or to indicate reverse only when paired reads

barcode  NONE  ccaac  V1F01V9RA
barcode  NONE  ccaac  V1F02V9RA
barcode  NONE  ccaac  V1F03V9RA
...

Linkers

The linker format is as follows:

linker yourLinkerString

example

linker tcag

Spacers

The spacer format is as follows:

spacer yourSpacerString

example

spacer tacgg

Ignoring samples with trim.seqs && trim.flows

You can set the group to “ignore” to indicate to mothur to cull them from the trimmmed file.

barcode    AATGGTAC    ignore
forward CCGTCAATTCMTTTRAGT  ignore

How different commands handle the *diffs parameters

trim.seqs

The trim.seqs command handles paired and single primers differently. This is designed to allow for the most flexibility in designing your analysis. Trim.seqs also sets a tdiffs (total differences) parameter equal to biffs+pdiffs, by default but can be adjusted. Let’s assume in all cases that pdiffs=2, bdiffs=1, tdiffs=3:

Paired primers are allowed 2 diff total between both the forward and reverse primers. With forward and reverse are each allowed two diffs. This can be a bit confusing so here’s an example:

forwardPrimer = 2 diffs, reversePrimer = 0 diffs -> primer will be successfully removed forwardPrimer = 2 diffs, reversePrimer = 1 diffs -> primer will not be removed because the paired primer total diffs = 3 forwardPrimer = 2 diffs, reversePrimer = 2 diffs -> primer will not be removed because the paired primer total diffs = 4 forwardPrimer = 1 diffs, reversePrimer = 0 diffs -> primer will be successfully removed forwardPrimer = 1 diffs, reversePrimer = 1 diffs -> primer will be successfully removed forwardPrimer = 0 diffs, reversePrimer = 1 diffs -> primer will be successfully removed forwardPrimer = 1 diffs, reversePrimer = 2 diffs -> primer will not be removed because the paired primer total diffs = 3
Separate forward and reverse primers handle the diffs differently. Both the forward and reverse primers are allowed 2 diffs each. Here’s the same example with separate primers.

forwardPrimer = 2 diffs, reversePrimer = 0 diffs -> primer will be successfully removed forwardPrimer = 2 diffs, reversePrimer = 1 diffs -> primer will be successfully removed forwardPrimer = 2 diffs, reversePrimer = 2 diffs -> primer will not be removed because the total diffs (bdiffs+pdiffs) = 4 forwardPrimer = 1 diffs, reversePrimer = 0 diffs -> primer will be successfully removed forwardPrimer = 1 diffs, reversePrimer = 1 diffs -> primer will be successfully removed forwardPrimer = 0 diffs, reversePrimer = 1 diffs -> primer will be successfully removed forwardPrimer = 1 diffs, reversePrimer = 2 diffs -> primer will be successfully removed

pcr.seqs

The pcr.seqs command handles paired and single primers the same. The pcr.seqs command allows you to set individual diffs values for the forward and reverse primers. This is designed to allow for maximum flexibility in removing primers. Let’s assume pdiffs=2, rdiffs=2, meaning allow 2 diffs in each primer.

forwardPrimer = 2 diffs, reversePrimer = 0 diffs -> primer will be successfully removed
forwardPrimer = 2 diffs, reversePrimer = 1 diffs ->  primer will be successfully removed
forwardPrimer = 2 diffs, reversePrimer = 2 diffs -> primer will be successfully removed
forwardPrimer = 1 diffs, reversePrimer = 0 diffs -> primer will be successfully removed
forwardPrimer = 1 diffs, reversePrimer = 1 diffs -> primer will be successfully removed
forwardPrimer = 0 diffs, reversePrimer = 1 diffs -> primer will be successfully removed
forwardPrimer = 1 diffs, reversePrimer = 2 diffs -> primer will be successfully removed

make.contigs

The make.contigs command is designed for paired primers, and operates like trim.seqs. Make.contigs also sets a tdiffs (total differences) parameter equal to biffs+pdiffs, by default, but can be adjusted. Let’s assume in all cases that pdiffs=2, bdiffs=1, tdiffs=3:

Paired primers are allowed 2 diff total between both the forward and reverse primers. With forward and reverse are each allowed two diffs. This can be a bit confusing so here’s an example:

forwardPrimer = 2 diffs, reversePrimer = 0 diffs -> primer will be successfully removed forwardPrimer = 2 diffs, reversePrimer = 1 diffs -> primer will not be removed because the paired primer total diffs = 3 forwardPrimer = 2 diffs, reversePrimer = 2 diffs -> primer will not be removed because the paired primer total diffs = 4 forwardPrimer = 1 diffs, reversePrimer = 0 diffs -> primer will be successfully removed forwardPrimer = 1 diffs, reversePrimer = 1 diffs -> primer will be successfully removed forwardPrimer = 0 diffs, reversePrimer = 1 diffs -> primer will be successfully removed forwardPrimer = 1 diffs, reversePrimer = 2 diffs -> primer will not be removed because the paired primer total diffs = 3