Illumina HiSeq

Model Organism: Worms Methods & Reagents
1

Hi all,

I am after some information regarding QC results for DNA libraries for WGS on an Illumina HiSeq.

My QC results after the PCR step in the library preparation are very low compared to human samples
prepared in the same batch. It looks as if the PCR step hasn’t worked very well. The lab sequencing
for me pretty much solely deals with human samples on their HiSeq and we are unsure if this is normal
with C. elegans DNA and you don’t need a high concentration
or if something has just gone wrong in the library preparation and we should start from scratch.

  1. What final concentration do people typically get for library preparations with C. elegans DNA?
    Conversely what is the lowest you can go outside illuminas recommendations?

  2. We used only 7 cycles of PCR (illumina recommends 10, but the lab here uses 7 for their human samples).
    Is this standard for C. elegans?

  3. What pM concentration do people use for cluster generation with C. elegans DNA?

Thanks everyone!

2

We’ve constructed dozens of worm libraries for Illumina sequencing, and low concentration is not usually a problem. Regarding your questions:

  1. Using the recommended 1 microgram of input gDNA, typical final concentrations are 20-50 ng/ul with 10 cycles of PCR. Three fewer cycles
    would yield 1/8 that amount.

  2. Ten cycles is standard for the Illumina kits.

  3. 8pM concentration for clustering on the HiSeq yields the maximum number of reads.

In our experience, low yields results from low amounts of input. Worm gDNA preps contain contaminants that absorb in the UV range, making
A260 measurements (such as Nanodrop) inaccurately high. Picogreen DNA fluorometry is unaffected by the contaminants.

Harold

3

Thanks Harold, much appreciated.