Miseq Amplicon sequencing library prep help

clmcmurray · 02-28-2014, 03:22 AM

Hey everyone,

I hope someone can help . I'm trying to preform amplicon sequencing on the Miseq. I'm using a custom target specific primer with the linker adapter to then follow the Illumina 16S library prepartion protocol. I'm struggling with the sensitivity of the inital PCR which isn't comparable with the level I managed to achieve with the target specific primer without the linker

. I need to improve the sensitivity as the samples I'm using have very low levels of DNA. Any suggestions on how to improve would be great.

shivabiochem · 03-17-2014, 11:19 PM

Hi,

Actually we prepared library for 16s for illumina miseq got good amount of amplification but we dint compare with the primer without the illumina tags. now i think from your question that we should once compare both.

bunce · 03-18-2014, 01:09 AM

dear clmcmurray,

Yes, when ever you use fusion-primers you suck efficiency out of the PCR and this can have a lot of impact on low-template samples. My advice would be to use standard and fusion primers and run both on qPCR (with sybr) to see how much of a 'shift' you are getting. And see how much input template you can put into your reaction before it becomes a problem (i.e you run out or it is inhibitory).

One option is to do the fusion PCR and spike in 10% of standard primer - this may help your PCR get off the ground (these products will not cluster downstream)

The last option is to use a ligation based approach to library build.

There is strengths and weaknesses to each of these approaches - best of luck.

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02-28-2014, 03:22 AM	#1
clmcmurray Junior Member Location: Birmingham, UK Join Date: Feb 2014 Posts: 1	Miseq Amplicon sequencing library prep help Hey everyone, I hope someone can help . I'm trying to preform amplicon sequencing on the Miseq. I'm using a custom target specific primer with the linker adapter to then follow the Illumina 16S library prepartion protocol. I'm struggling with the sensitivity of the inital PCR which isn't comparable with the level I managed to achieve with the target specific primer without the linker . I need to improve the sensitivity as the samples I'm using have very low levels of DNA. Any suggestions on how to improve would be great.

03-17-2014, 11:19 PM	#2
shivabiochem Junior Member Location: INDIA Join Date: Feb 2014 Posts: 1	Hi, Actually we prepared library for 16s for illumina miseq got good amount of amplification but we dint compare with the primer without the illumina tags. now i think from your question that we should once compare both.

03-18-2014, 01:09 AM	#3
bunce Member Location: Perth Join Date: Sep 2012 Posts: 55	dear clmcmurray, Yes, when ever you use fusion-primers you suck efficiency out of the PCR and this can have a lot of impact on low-template samples. My advice would be to use standard and fusion primers and run both on qPCR (with sybr) to see how much of a 'shift' you are getting. And see how much input template you can put into your reaction before it becomes a problem (i.e you run out or it is inhibitory). One option is to do the fusion PCR and spike in 10% of standard primer - this may help your PCR get off the ground (these products will not cluster downstream) The last option is to use a ligation based approach to library build. There is strengths and weaknesses to each of these approaches - best of luck.