Chemistry and protocol differences in Miseq, GAII and Hiseq?

dwang · 01-12-2012, 10:20 AM

Does anyone know any chemistry and protocol differences in Miseq, GAII and Hiseq? My amplicon-enriched libraries worked well in GAII and HIseq but not in Miseq in terms of mappable reads as well as amplification uniformity. They are same libraries using same barcodes and sequencing primers.

Thanks a lot

Karel · 02-19-2013, 08:29 AM

I wouldnt mind having an answer on this as well.

GenoMax · 02-19-2013, 09:07 AM

MiSeq has known problems with libraries that have low nucleotide diversity. There are several threads (an example thread: http://seqanswers.com/forums/showthread.php?t=27563) on SeqAnswers that address that issue. There are workarounds proposed but no definite solution as yet.

As for the answers regarding the chemistry used for the 3 platform and the differences search through these FAQ's.

kwaraska · 02-19-2013, 09:49 AM

Also, the Tm for custom primers are different between HiSeq and MiSeq.

The HiSeq custom primer needs only to be above 56, where MiSeq must be above 66. It has to do with the difference in the annealing of the primer. We found that out the hard way!

Also, legacy (non-TruSeq) Single read libraries will not run on MiSeq. Another item we found out the hard way!

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01-12-2012, 10:20 AM	#1
dwang Junior Member Location: USA Join Date: Apr 2010 Posts: 1	Chemistry and protocol differences in Miseq, GAII and Hiseq? Does anyone know any chemistry and protocol differences in Miseq, GAII and Hiseq? My amplicon-enriched libraries worked well in GAII and HIseq but not in Miseq in terms of mappable reads as well as amplification uniformity. They are same libraries using same barcodes and sequencing primers. Thanks a lot

02-19-2013, 08:29 AM	#2
Karel Junior Member Location: Netherlands Join Date: Feb 2013 Posts: 1	I wouldnt mind having an answer on this as well.

02-19-2013, 09:07 AM	#3
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,080	MiSeq has known problems with libraries that have low nucleotide diversity. There are several threads (an example thread: http://seqanswers.com/forums/showthread.php?t=27563) on SeqAnswers that address that issue. There are workarounds proposed but no definite solution as yet. As for the answers regarding the chemistry used for the 3 platform and the differences search through these FAQ's.

02-19-2013, 09:49 AM	#4
kwaraska Senior Member Location: Boston,MA Join Date: Nov 2008 Posts: 122	Also, the Tm for custom primers are different between HiSeq and MiSeq. The HiSeq custom primer needs only to be above 56, where MiSeq must be above 66. It has to do with the difference in the annealing of the primer. We found that out the hard way! Also, legacy (non-TruSeq) Single read libraries will not run on MiSeq. Another item we found out the hard way!