ssDNA - what happens during sequencing?

[Kerensa]

Hi Guys,

I've got some Illumina sequencing data from genomic bacterial DNA, however I have reason to believe that my original DNA sample may have contained some single stranded DNA (from a phage).

My question is, will this DNA also have been sequenced? What happens to ssDNA during library prep?

I understand that there is a step after nebulization, which 'fills in' single stranded overhangs. Will this also fill in fragments that are totally single stranded? Or does the T4 polymerase need a double stranded section to initiate filling in?

Thanks for your help,

Kerensa.

[ECO]

T4 Pol (or klenow exo- used for A-tailing) will not prime de novo to double strand a completely single stranded fragment, however could fill in _mostly_ ssDNA fragments.

ssDNA is one of those contaminants that can contribute to falsely high OD readings, and thus incorrect quantitation of one's library. It will happily persist through most purification methods assuming it's large enough to not be removed by a size selection step. If you QC your final library with bioanalyzer or qPCR you should be good to go.

[ScottC]

You might get some single strands being sequenced; There's a couple of publications where strand-specific RNA sequencing was carried out by producing cDNA, omitting the second strand synthesis, and proceding with sequencing. However, it may rely on various double stranded products in the mix such as DNA-RNA hybrids and so forth... Just something to consider.

PLoS Genet. 2009 Jul;5(7):e1000569. Epub 2009 Jul 17.
A strand-specific RNA-Seq analysis of the transcriptome of the typhoid bacillus Salmonella typhi.

http://www.ncbi.nlm.nih.gov/pubmed/19609351

Nucleic Acids Res. 2009 37(22): e148.
A simple method for directional transcriptome sequencing using Illumina technology

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2794173/