dual index oligos

Commands in mothur
1

I’m trying to switch to dual index sequencing from single index Caporaso primers. I’m still using the Caporaso primers as the reverse (p7, i7, pad linker, 806R) and am using Kozich v4.SA501-508. I wanted to have the MiSeq demultiplex the sequences but set up the sample sheet incorrectly (didn’t rc i7) so now I’m trying to use make.contigs and am doing something wrong because all my seqs are ending up in the scrap.


my oligos is

barcode rev.comp.i7 i5 sample.name

barcode GGAGACAA ATCGTACG n08
barcode GGAGACAA ACTATCTG n07
barcode GGAGACAA TAGCGAGT n18
barcode GGAGACAA CTGCGTGT n20
barcode AAGGAGCG TCATCGAG n27
barcode AAGGAGCG CGTGAGTG n34
barcode AAGGAGCG GGATATCT n41
barcode AAGGAGCG GACACCGT n45

I’ve tried checkorient=t and still everything ends up in scrap.

2

I’m sure this is a really tiny error but I just can’t figure it out.

looking at Kozich-I1 should be the reverse compliment of i7 correct and I2 is i5? then the oligos should be set up

barcode rev.comp.i7 i5 samplename

Or should it be

barcode i7 i5 samplename

3

Finally figured this out. You need to trim the last 4 bases of the index after you reverse compliment. I was trimming the last 4 bases then reverse complimenting-doh