ddRAD/GBS lib conc on HiSeq 3000/4000 ?

[BioGenomics]

Hi all,

Has anybody any advice on the "best" concentration (pM) to load a ddRAD or GBS libraries on a HiSeq 3000/4000 ? ddRAD have 300-600 bp, while GBS more around 250-350 bp lib sizes. I know the longer fragments of ddRAD might create a problem may be with the new chemistry/patterned flowcells.

Also 10% Phix would be ok ?

Cheers,

Greg

[ATϟGC]

Hi,

The optimal loading concentration and phiX % spike in will likely depend largely on the nucleotide sequence diversity at the start of your reads (on the HiSeq 2000 the first 12 bp is the most critical I think). Depending on how your ddRAD libraries were prepared they may be quite diverse (i.e. balanced proportions of each nucleotide at each position) or suffer from low sequence diversity. From what I read (http://support.illumina.com/sequenci...questions.html) the HiSeq 4000 still has low-diversity issues so it would be helpful if you could provide information as to how diverse the start of your reads are. A matrix of the nucleotide diversity for the first 12 bp like the one in the last post of the link below might help others give you recommendations.

http://seqanswers.com/forums/showthread.php?t=66196&page=2

After having some run failures last year due to supposed nucleotide diversity issues on a hiseq 2000, we nearly perfect runs by adding some staggered barcodes to break up the restriction site, included a 15% phiX spike in, and clustered at 600-700K/mm^2.