Demultiplexing dual indexed libraries

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Lilnet

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Nov 21, 2018, 8:25:57 PM11/21/18

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Hi everyone,

would like to ask if anyone has had experience in demultiplexing dual indexed libraries?

The header of the output file, below, is where the red highlighted area is the dual indexed primer sequence, and I also have inline barcodes within the sequenced reads.

So far while demultiplexing we have tried including only the barcode sequences, both indexes, and only one index, but these have not allowed us to demultiplex the libraries appropriately.

Would be happy to learn if anyone has tried demultiplexing such libraries.

Thank you!

@E00582:207:HVC3WCCXY:6:1101:9211:1731 1:N:0:ATCACGAT+AGATCTCG

NACCAAATTCACGTAAACATCAAGATGAATGAGAATCCATATTGGTTTGAAATCAATTTATCTTACTTTTTCTTCAGTTTATTTGGAGTTTTTTTAATTCTTCTAAAGTACAGAAGGAGTATGTTTGTGTATTTCTTATAGGAAAAAAAC

#A<AFJJFJJFJFJFJF<JJFJFJJ-FJJJJJFJJJ-FJJJJJAJF-F7JJJJ<JJJJFJJJJF-FJJJAJFJ<<FAJJJFFA<JJ7A<AF<JFJJFJ-A-FJJJJJJJ<F-A<FJFJJ--77FJAA-77--<JA-7AA-<-7AF--777

@E00582:207:HVC3WCCXY:6:1101:9252:1731 1:N:0:ATCACGAT+AGATCTCG

NGATCAATTCAATAATGAGTGAAATGTCAGGAACAAAACACACGTAGCTTTTCGAATAGATGTTGTCTGATACGAAGGAAAGCAGAAAAGAAACGAAGCTGTTAAACATTTAGATTTTTTTGTTTACTAAAAGAGTTATTACTTAATAGA

#A-AFJJFJJJJJJJFAJ7FJJJJJFJJJAJJJFJJJJJJFJJ<FJFJ-F-FFFFJJJ7-FFJJ<FJFJ---FA-<AFJJAF-<FFFF<FJJJJJAFJ-<FJJJJJJJJ<J7FJAF7FF-7F7AJJ7AA7FJ<J---A7-A---7-7FJJ

@E00582:207:HVC3WCCXY:6:1101:10490:1731 1:N:0:ATCACGAT+AGATCTCG

NCGGTAATTCAGTAAAACTTATTTGGAGCTAATACAGAAATGTTTAGAAGGCTACATCAAGAGACAAGTTTGTTCAACAGAGTATATGTATAACCAAGTGTATTCACTGTATCACATGATGGTCGTAAGGTGTATAAATACGGCAGGGGT

#AAFFJJJJJJJJJFJJJ<JJJJJJJJJJJJJJJJJJJJ<AJJFJJJJ<JAFJJJJJJJJJJJJ7JJJJJJJJFJJJJJJFJ-FFJJJ-FFJJJJJFJ-FJJJJJJF7JJJFJJ<FAJJJFJ-FJA7AFJ-AAFJ<JA<A<A-77-7-7-

@E00582:207:HVC3WCCXY:6:1101:10795:1731 1:N:0:ATCACGAT+AGATCTCG

NCACAAAAACAAGATGTTTCATTAGGTAGAAGAAAGAAATTGGAGATTTCAGACCAAAGAGTTCTGTGAAAGAATTATAACAATAAGTTTCATCATATTTTGTCTGTATGCTTGTCTAGAAAAGTCGCTTACTTAGATGAAATATTTGTT

#AAFFJJJJJJJJJAJJFJJJJJJJJJJJJJJJJJJJJJJ<JJJJJJJ<FJFJ-FJJJJFJ<JJAJFJFFJJJJJJJJJJFJJJJJJ-7JJAJFJJJJ-FJJJJJJJJJJJJJJ<JAJFJ<JJJJ7FA-<FFFJJAJ<AFJF-FF<<FJ-

@E00582:207:HVC3WCCXY:6:1101:11891:1731 1:N:0:ATCACGAT+AGATCTCG

NCACAAATTCACCAAAGAACATCGTGCTATAGGGTTTGAAAATATCTTGAAAGAAAAGCTGTTATAACAATTGTAATTAAAGGGTTGATTCTTGGCTTGCTTGTCCTGAAAATGCTTCTGACTGCATAAAGCTATAGGATACTTTATCAA

#<AAFJJJJJJJJJJJJJJJJJFJFFJ<F7JJJJJJ<JJJJJJJJJFJ<JJJJJJJJJAJJJJJJJJJJJ<JJFFJJJJJFJFJJFJF-JJJJJJFJF-FJJJJJJJJJFJJJJ-FJJFJ7F7FJFJAFJ--7FJ-7-7-A<--F-7-FA

Catchen, Julian

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Nov 21, 2018, 9:46:48 PM11/21/18

to stacks...@googlegroups.com, Lilnet

Hi,

Please tell us what your raw, multiplexed files currently look like and
what type of demultiplexed output you want. Also, please give us the
command you have tried and what the result was.

Lilnet wrote on 11/21/18 7:25 PM:

> Hi everyone,
>
> would like to ask if anyone has had experience in demultiplexing dual
> indexed libraries?
>
> The header of the output file, below, is where the red highlighted area
> is the dual indexed primer sequence, and I also have inline barcodes
> within the sequenced reads.
> So far while demultiplexing we have tried including only the barcode
> sequences, both indexes, and only one index, but these have not allowed
> us to demultiplex the libraries appropriately.
> Would be happy to learn if anyone has tried demultiplexing such libraries.
>
> Thank you!
>

> @E00582:207:HVC3WCCXY:6:1101:9211:1731 1:N:0:*ATCACGAT+AGATCTCG*

>
> NACCAAATTCACGTAAACATCAAGATGAATGAGAATCCATATTGGTTTGAAATCAATTTATCTTACTTTTTCTTCAGTTTATTTGGAGTTTTTTTAATTCTTCTAAAGTACAGAAGGAGTATGTTTGTGTATTTCTTATAGGAAAAAAAC
>
> +
>
> #A<AFJJFJJFJFJFJF<JJFJFJJ-FJJJJJFJJJ-FJJJJJAJF-F7JJJJ<JJJJFJJJJF-FJJJAJFJ<<FAJJJFFA<JJ7A<AF<JFJJFJ-A-FJJJJJJJ<F-A<FJFJJ--77FJAA-77--<JA-7AA-<-7AF--777
>

> @E00582:207:HVC3WCCXY:6:1101:9252:1731 1:N:0:*ATCACGAT+AGATCTCG*

>
> NGATCAATTCAATAATGAGTGAAATGTCAGGAACAAAACACACGTAGCTTTTCGAATAGATGTTGTCTGATACGAAGGAAAGCAGAAAAGAAACGAAGCTGTTAAACATTTAGATTTTTTTGTTTACTAAAAGAGTTATTACTTAATAGA
>
> +
>
> #A-AFJJFJJJJJJJFAJ7FJJJJJFJJJAJJJFJJJJJJFJJ<FJFJ-F-FFFFJJJ7-FFJJ<FJFJ---FA-<AFJJAF-<FFFF<FJJJJJAFJ-<FJJJJJJJJ<J7FJAF7FF-7F7AJJ7AA7FJ<J---A7-A---7-7FJJ
>

> @E00582:207:HVC3WCCXY:6:1101:10490:1731 1:N:0:*ATCACGAT+AGATCTCG*

>
> NCGGTAATTCAGTAAAACTTATTTGGAGCTAATACAGAAATGTTTAGAAGGCTACATCAAGAGACAAGTTTGTTCAACAGAGTATATGTATAACCAAGTGTATTCACTGTATCACATGATGGTCGTAAGGTGTATAAATACGGCAGGGGT
>
> +
>
> #AAFFJJJJJJJJJFJJJ<JJJJJJJJJJJJJJJJJJJJ<AJJFJJJJ<JAFJJJJJJJJJJJJ7JJJJJJJJFJJJJJJFJ-FFJJJ-FFJJJJJFJ-FJJJJJJF7JJJFJJ<FAJJJFJ-FJA7AFJ-AAFJ<JA<A<A-77-7-7-
>

> @E00582:207:HVC3WCCXY:6:1101:10795:1731 1:N:0:*ATCACGAT+AGATCTCG*

>
> NCACAAAAACAAGATGTTTCATTAGGTAGAAGAAAGAAATTGGAGATTTCAGACCAAAGAGTTCTGTGAAAGAATTATAACAATAAGTTTCATCATATTTTGTCTGTATGCTTGTCTAGAAAAGTCGCTTACTTAGATGAAATATTTGTT
>
> +
>
> #AAFFJJJJJJJJJAJJFJJJJJJJJJJJJJJJJJJJJJJ<JJJJJJJ<FJFJ-FJJJJFJ<JJAJFJFFJJJJJJJJJJFJJJJJJ-7JJAJFJJJJ-FJJJJJJJJJJJJJJ<JAJFJ<JJJJ7FA-<FFFJJAJ<AFJF-FF<<FJ-
>