SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
How to get the highest accuracy with BWA for metagenomics RickBioinf Bioinformatics 0 12-24-2012 04:03 AM
Homopolymers // ValleyFilters Splinter479 Bioinformatics 1 08-16-2012 02:40 AM
Ion torrent blog on raw accuracy - good primer for accuracy grand challenge lek2k Ion Torrent 3 09-20-2011 12:13 AM
454 accuracy with homopolymers dina Bioinformatics 3 07-30-2010 04:13 AM
Accuracy of SOLiD platform dgmacarthur SOLiD 14 09-11-2008 02:07 AM

Reply
 
Thread Tools
Old 10-24-2014, 12:54 AM   #1
simon2
Junior Member
 
Location: Germany

Join Date: Oct 2014
Posts: 2
Smile Which platform provides the highest accuracy on homopolymers?

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
simon2 is offline   Reply With Quote
Old 10-24-2014, 01:21 AM   #2
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,234
Default

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.
nucacidhunter is offline   Reply With Quote
Old 10-24-2014, 01:34 AM   #3
simon2
Junior Member
 
Location: Germany

Join Date: Oct 2014
Posts: 2
Default

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.
simon2 is offline   Reply With Quote
Old 10-24-2014, 02:02 AM   #4
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,234
Default

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.
nucacidhunter is offline   Reply With Quote
Reply

Tags
accuracy, homopolymers

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 04:46 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO