Only one barcode present after dual indexing

[k_a_r_o_l]

Hi,

I have such a problem. I had my MiSeq experiment where I was sequencing some amplicons which were dual indexed (each sample had 2 unique barcodes)

Something like this:
sample1 - AB
sample2 - CD
sample3 - EF

So we didnt have any AD, CF, AF... ect.

In the end, MiSeq could not demultiplex our reads because only one barcode was detected.

Something like this:

@HWI-M02221:13:000000000-A7W2J:1:1101:11982:1825 1:N:0:NGNNNCTNCGTCTAAT
ACTGTCTTCACGCAGAAAGCGTCTAGCCATGGCGTTAGTACGAGTGTCGTGCAGCCTCCAGGCCCCCCCCTCCCGGGAGAGCCATAGTGGTCTTCGGAACCGGTGAGTACACCGGAATCGCTGGGGTGACCGGGTCCTTTCTTGGAACAACCCGCTCAATACCCAGAAATTTTGGCGTGCCCCCGCGAGATCACTAGCCGAGTATTGTTTGTTCCCCAAAGGCCTTGTGGTACTGCCTCATATNNTNCTTGGNATCTTAATATCACCCTTTNTGACTCTATNCCNCGAGGCTGGTCTCCN

@HWI-M02221:13:000000000-A7W2J:1:1101:19763:1825 1:N:0:NCNNNCTNCTACTGAC
ACTGTCTTCACGCAGAAAGCGTCTAGCCATGGCGTTAGTACGAGTGTCGTGCAGCCTCCAGGACCCCCCCTCCCGGGAGAGCCATAGTGGTCTGCGGAACCGGTGAGTACACCGGAATCGCTGGGTTGACCGGGTCCTTTCTTGGAACAACCCGCTCAATACCCAGAAATTTGGGCGTGCCCCCGCGATATCACTATCCTCGTAGTCTTGGGTCGCGAAACGCCTTTTGTTACTGCCTTATACNNTNCTTTANACCTTAATCTCACACTTTNTGACTCCATNCCNACTCGCTAACCTCCN


Do you know any method to demulitpex such kind of data, considering that even with one barcode I know from which sample originates my amplicon. Any software, sugestions?

[GenoMax]

You may need to do this offline (I am not sure if you can run MiSeq reporter asking it to ignore first barcode and use only the second or use BaseSpace, someone else can comment on that).

You will need access to a linux machine and a software called bcl2fastq that you can download from illumina. If you are not IT savvy the you will need to get help from someone. A specific option will need to be provided to the configureBclToFastq program (--use-bases-mask Y*,n*,I6n,Y*) when you do the demultiplexing.

This will only work if the second barcode is unique!

[k_a_r_o_l]

I am sure that second barcode is unique. Thank You, I will try this.

[kmcarr]

A specific option will need to be provided to the configureBclToFastq program (--use-bases-mask Y*,n*,I6n,Y*) when you do the demultiplexing.

Actually, since your barcodes are 8 bases long and you want to use the entire second barcode you should use:

--use-bases-mask "Y*,n*,I*,Y*"

Be sure to enclose that pattern in quotes as shown otherwise your command line interpreter will try to glob the '*'.