Quality Control of Solexa

[aquleaf]

Hi, we are carrying out mRNA-seq on Solexa. My questions are:

1, How to know whether the run succeeds or not?

2, What's the quality criteria for good reads?

One unrelated question, does sequencing depth mean sequencing coverage?

Thanks.

[simonandrews]

Hi, we are carrying out mRNA-seq on Solexa. My questions are:

1, How to know whether the run succeeds or not?

Generally the diagnostics on the sequencer itself will tell you if the sequencing worked OK. Most centres will run some kind of control sample on each flowcell (either as a dedicated lane, or spiked in with each library), and this should work even if there is a problem with the individual libraries. In our experience Illumina runs tend to either work really well, or fail completely (and failures are pretty rare).

We find that library failures are much more common than machine failures, so doing some additional QC on your library is definitely worthwhile.

2, What's the quality criteria for good reads?

There aren't fixed parameters for what counts as good quality data. In the QC software we produce we've set warning and failure limits for a variety of different parameters based on looking at loads of data coming through our facility, but these are intended as rules of thumb rather than hard cutoffs. Running some additional QC on your libraries will definitely give you a better impression of how well your runs went.

[DNautomate]

Hi,

I would like to hear what people are doing to assess library quality before submission to a Solexa sequencer other than running a Bioanalyzer read? qPCR?

Thanks,

[vtosha]

Manual recommends qPCR as main QC step but our last run had a problem: we use qPCR data for quantity of libraries for one lane but number of clusters was a only a quarter from control phiX (standards for qPCR was the same phiX). Techsupport can not answer for our question about it.