Read length for Illumina mate pairs

[Linnea]

Hey all,

I've heard that with the new, longer Illumina reads (>100bp), for mate-pair sequencing the amount of chimeric reads has increased strikingly. (Because it is much more likely to read over the junction). Is this true? Anyone having experience from this?

I've searched for information, but the only thing I found was a quite old post saying that Illumina doesn't recommend longer read length than 36bp for mate-pair sequencing.

We are about to sequence one full flowcell of Illumina mate-pairs, and the default read length is now 109bp - will this work or is it better to choose a shorter read length, like 36 or 50bp?

Any advice would be appreciated!

[kmcarr]

Illumina has not changed its recommendation for read lengths of mate-pair libraries. From the MatePair_v2_2-5kb_SamplePrep_Guide_15008135_A:

When sequencing a mate pair library, Illumina recommends a read length no
longer than 36 bases. A longer read length elevates error rates, because
longer reads are more likely to cross over the junction of the two joined ends
of a size-selected fragment. The Illumina analysis pipeline discards these
junction reads, since they do not align to the reference sequence.

While the Illumina platform is capable of generating longer reads they are not appropriate for every application. Mate-pair data is not meant to provide large amounts of coverage. Coverage should be generated with long reads from paired end (or single end) libraries. A small amount of mate-pair data is then combined with these to order contigs into scaffolds.

What are you sequencing? Is it de novo? (I would assume so if you are doing mate pair.) Do you have any other sequence data you will be combining with this?

[Linnea]

Thanks for the reply!

I hadn't seen that SamplePrep guide. Then we'll go for 36bp!

Yes, we're doing de novo and we allready have two full flowcells sequenced with paired-ends, so the current assembly has a coverage of ~40X. The mate-pair flowcell is just for scaffolding so I guess the coverage of that isn't too important.

Cheers,
Linnéa