Demultiplexing illumina reads

[Bioinform]

Hi there,

I have some basic questions. It would be really great if any one could help me out.

1. I wanted to know different ways of demultiplexing. I have worked with demultiplex.pl script targeting barcodes.
Is there any scripts or ways targeting sequence.
I want both the F and R reads in one file the one i am using gives me seperate file (demultiplex.pl).

2. After demultiplexing i get different number of reads in F and R files (i suppose it to be equal in number). What would be the threshold of difference in the number of reads in F and R files?

Thanks

[GenoMax]

I assume these are in-line barcodes because otherwise illumina's software takes care of demultiplexing. Not sure why you are getting unequal number of F/R reads.

Is there a specific reason you want F/R reads in one file?

[JackieBadger]

You shouldn't have different numbers of reads in R1 and R2 files. Maybe your demultiplex script is throwing some sequences out based on quality or Ns?

Are R1 and R2 derived from a targeted amplicon, and should they overlap?
If so you should stitch them together first. Then you can use jMHC to demultiplex (after conversion to fast).

A little more information on your system would help in getting a clearer answer.

[Bioinform]

Hi Guys thanks for the quick reeply.

GenoMax

my demultiplex script is not giving me equal number of reads, they pull F and R in different file so they may not be equal.
I think there are demultiplex script that pulls both the reads in one file, pulls F and automatically pulls R, so that the F and R reads become paired.

JackieBadger
I was guessing so too.

I have miseq data. I pooled 8 libraries and send them for sequencing. Since the barcodes are not the illumina kit barcodes i told them to send the files and i would demultiplex them.

With CLC i get similar number of F and R reads (paired in one file).
But with the demultiplex.pl script i get different number of F and R reads separately.
I was looking for demultiplex script that pulls both the reads in one file, pulls F and automatically pulls R targeting the sequence, and F and R reads get paired.

Thanks

[Bioinform]

Hi,

Does anyone has perl or pythogn script that can pull out Reads (Forward) from R1 file and corresponding pair (Reverse) from R2 file. CLC workbench does this but want some script that works alike.

Many thanks