Illumina Read Names: /2 Vs. /3

Question

[Newvin]

The sequencing core at my university performed paired end RNA-seq on some of our lab's samples using Illumina sequencing technology. My understanding is that generally the forward and reverse read names are designated with trailing /1 and /2 e.g.

D5KHLFN1_0181:1:1101:1209:2028#0/1

D5KHLFN1_0181:1:1101:1209:2028#0/2

However, our results came back with /1 and /3 suffixes instead. The sequencing core claims this is an artifact of "tru-seq" sequencing. I was wondering if anyone could confirm this and/or elaborate.

(The main reason for my interest here is that certain de novo transcriptome assemblers actually require the "/2" rather than "/3", forcing me to do a sed search/replace)

Thanks!

Answer 1

[Ido Tamir]

I expand on my previous answer on request and also push illumina2bam.

Illumina allows combining multiple libraries into one lane using multiplexing. Illumina multiplexes with an additional read that reads a short sequence within in the adapter after it has read the first read. This results in a sequence of read1:index-read for single end reads and read1:index-read:read2 for paired end reads. So paired end read2 -> /3. Seems like in your case an indexed read was specified - maybe it was necessary for other lanes, or the wrong program was chosen.

It is superior to simply adding a short barcode at the beginning of the product because you have less problems with basecalling (normal complexity at start of reads).

I suggest everyone involved in collecting data from the machine to have a look at bam as primary output format instead of fastq and maybe push for it:

1. you have less problems with the scale of the quality values. This was changed 4 times now.
2. more important: all the provenance information is saved within the file, and if you have a correctly working pipeline set up - I am far from that :-( - all programs save the transformations on the data in the file. You know exactly what happened (which parameters, which version etc...).

2 possibilities exist to my knowledge:

* [illumina2bam] which reads directly from the saved bcl files and its **easy** to use!
* [IlluminaBasecallsToSam] picards which I think starts from the qseq files.

In the case of illumina2bam there is a great pipeline that takes the basecalls and puts the index read into the tags of the read in the bam file. Easy to parse, easy to split, merge etc.

[Ido Tamir]

I rewrote the answer. I started with my agenda in promoting bam.

[Bach]

Errrrm ... I did not get that. Care to explain a bit more in-depth?

[Bach]

Thank you bery much for that ... I learned something really new and valuable today. Although I am not sure I like the BAM idea. FASTQ is nice because one can do a lot of tricks already on the command line with head, tail, sed, etc.pp and that is not possible anymore with BAM.

[Bach]

Thank you very much for that ... I learned something really new and valuable today.

Although I am not sure I like the BAM idea. FASTQ is nice because one can do a lot of tricks already on the command line with head, tail, sed, etc.pp and that is not possible anymore with BAM.

Answer 2

[User 1244]

I had the same issue, I simply used sed to convert my reads to /2 and moved on. Sequencing artifact is absurd. This is explained better in the previous answer.