Library fragment size

[tahneesaunders]

Hey All,

I have previously done my own library prep for PE100 on HiSeq4000 and my fragment size is around 300bp recommended.

This time when I completed library prep my fragments range from 300-400bp and have failed QC done by the sequencing core.

Some samples have a tiny peak at 113-117bp - the library prep kit I use is NEBnext RNA Ultra Library prep with ribosomal RNA depletion - this kit says that peaks at 80bp and 125bp is an issue and requires clean up.

The sequencing core did a clean up but some samples are still failing. I have written to them to ask exactly what QC parameters are failing.

Bioanalyser traces attached if that helps.

Sample 4-6 were from difficult to handle samples so it isn't a surprise that library prep didn't work.
Sample 23-24 are blank controls.

I guess my main questions are:
1) Have you successfully sequencing PE100 with fragment sizes up to 400bp?
2) Is the small peak at 113-117bp an issue?

Any suggestions / comments are welcome!

Thank you!

[pmiguel]

1) We routinely sequence libraries with tails stretching >400bp, sometimes to 1-2kb. But we use a HiSeq 2500. From what I've read, longer inserts can be an issue for HiSeq 3000/4000 because these amplicons can "jump the walls" of the patterned flowcell, creating duplicate (and possibly mixed) clusters.
But I don't know if, in practice, this really is an issue.
2) Some percentage of your reads will derive from adapter-dimers (zero insert amplicons.) The percentage of these reads is always higher than expected based on relative molar concentrations. Alas, if the concentration of the adapter-dimer is fairly high, you can't get rid of all of it using gel cuts or ampure -- because some of it will be annealed to full-insert library molecules since they share the same adapter sequences.

--
Phillip