Which platform provides the highest accuracy on homopolymers?

[simon2]

Hi,

what NGS platform provides the highest accuracy regarding length and composition of homopolymeric stretches (e.g. (TTT)10) as being found for example in repetitive DNA elements?

I read that 454 apparently does not work so well. We already tried out Illumina MiSeq and we were not satisfied. What about SOLiD? Can you share any experience?

Thank you
Simon

[nucacidhunter]

If the issue arises from PCR step with polymerase not being able to efficiently synthesise complementary strands in repeat region, then SOLiD is not going to be good either. Illumina is good at sequencing step through repeat region because it incorporates nucleotides one at a time due to terminator which prevents multiple incorporation once one nucleotide pairs. 454 and Ion are the worst because in addition to PCR issue they cannot control the number of nucleotides that are paired with complementary bases. They flood the Picotitre plate or Chip with one nucleotide at a time and if homopolymer length is higher than 5, normally the signal is saturated. If you are sequencing a genome, Illumina's PCR free library prep kit would give good results.

[simon2]

Thank you very much.

I observed that T-stretch-length is often overestimated in my Illumina MiSeq runs (e.g. true length is 28, but the read says its 30). According to Whiteford et al. 2009 T signals accumulate over cycle due to incomplete fluorophore cleavage.

So I think it is rather an imaging problem than a PCR problem. Although phasing might also be a problem, I observed that the nucleotides following the T-stretch are overlayed by T's and not shifted.

[nucacidhunter]

Since 2009 Illumina chemistry has changed several times, I do not know if the issue that referred in Whiteford et al. 2009 has been addressed or not. If T signal is the issue, one workaround might be having short inserts to the size of one read sequencing cycle. So, in paired end sequencing Ts will be replaced with As.