Short PCR product sequencing with Miseq

vl80 · 11-10-2012, 05:36 PM

Is it possible to omit the fragmentation step of the library preperation, proceed with the rest of the steps and sequence PCR products of 90 - 450 bp using Miseq?
I am just theorising as I have no prior experience in this but need to plan a way to sequence short PCR products woth Miseq. Any inputs are welcome.
Thank you

JackieBadger · 11-10-2012, 06:01 PM

without knowing what your library prep is....MiSeq is spec'd to sequence 2x250base. So if your total product including adapters etc is below 500 bases you can sequence your product using paired ends and stitch together the over lapping sequences.
you can in theory sequence any size fragment to to the total possible.

vl80 · 11-10-2012, 06:13 PM

Thank you very much for the reply. I wonder if anyone has attempted this modification before and if there are any publications? I tried to search for it but was not successful.

JackieBadger · 11-11-2012, 04:22 AM

what modification?
Nextera library prep routinely sequences short fragments.
Just get your PCR product and run it through the TrueSeq library prep and sequence it.
Like I said, there are many ways to prepare a library for sequencing.

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
Bioruptor shearing of PCR product	bernieball	Sample Prep / Library Generation	1	11-25-2013 07:37 AM
Sequencing Short PCR Products	Dynamac	Sanger/Dye Terminator	4	07-23-2012 10:27 AM
PCR product normalization	Palecomic	Sample Prep / Library Generation	0	01-20-2011 02:50 AM
Excess PCR primers in final product	PaulineF	Illumina/Solexa	19	07-27-2010 05:23 AM
Request for sequencing of PCR product	Damann	Core Facilities	0	11-05-2009 08:34 AM

11-10-2012, 05:36 PM	#1
vl80 Member Location: Asia Join Date: Nov 2012 Posts: 34	Short PCR product sequencing with Miseq Is it possible to omit the fragmentation step of the library preperation, proceed with the rest of the steps and sequence PCR products of 90 - 450 bp using Miseq? I am just theorising as I have no prior experience in this but need to plan a way to sequence short PCR products woth Miseq. Any inputs are welcome. Thank you

11-10-2012, 06:01 PM	#2
JackieBadger Senior Member Location: Halifax, Nova Scotia Join Date: Mar 2009 Posts: 381	without knowing what your library prep is....MiSeq is spec'd to sequence 2x250base. So if your total product including adapters etc is below 500 bases you can sequence your product using paired ends and stitch together the over lapping sequences. you can in theory sequence any size fragment to to the total possible.

11-10-2012, 06:13 PM	#3
vl80 Member Location: Asia Join Date: Nov 2012 Posts: 34	Thank you very much for the reply. I wonder if anyone has attempted this modification before and if there are any publications? I tried to search for it but was not successful.

11-11-2012, 04:22 AM	#4
JackieBadger Senior Member Location: Halifax, Nova Scotia Join Date: Mar 2009 Posts: 381	what modification? Nextera library prep routinely sequences short fragments. Just get your PCR product and run it through the TrueSeq library prep and sequence it. Like I said, there are many ways to prepare a library for sequencing.