TrueSeq PCR free vs Nextera Mitogenome seq

frascom · 05-22-2019, 10:54 AM

I wondered if anyone had any recommendations for TrueSeq PCR free kits vs Nextera XT for mitogenome sequencing around 17,000bp pooling PCR fragments and around 192 individual samples on a single Miseq run?

Are there benefits of considering one over the other or protocols to follow for sample size and barcoding

thanks for the help

SNPsaurus · 05-22-2019, 01:47 PM

Can you multiplex that much on a Miseq and get enough reads per sample to reconstruct the mitogenome? I guess if the nuclear genome isn't large then a reasonable fraction of reads will go to the mitogenome.

You can use partial reaction of just Nextera DNA Flex and get good results. That might be the cheapest route.

frascom · 05-23-2019, 06:02 AM

I have amplified the mitogenome separately so there should no concern of reads. The main aim is trying to save some costs due to barcoding 192 individuals whilst achieving a high quality of reads. The benefit of NexteraXT I think is the availability of barcodes index for up to 384 samples. Although another option would be to use propriety barcodes so save a lot of money

SNPsaurus · 05-23-2019, 12:11 PM

I see, that makes sense for amplified mtDNA.

You can combine the Nextera Flex and Nextera XT barcode sets for Nextera Flex preps. We do that for increased multiplexing.

nucacidhunter · 05-23-2019, 08:25 PM

plexWell 96 or 384 could fit your library prep. It seems to be cheap and also quick.

https://seqwell.com/product/pw96/

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05-22-2019, 10:54 AM	#1
frascom Junior Member Location: Kansas Join Date: May 2019 Posts: 4	TrueSeq PCR free vs Nextera Mitogenome seq I wondered if anyone had any recommendations for TrueSeq PCR free kits vs Nextera XT for mitogenome sequencing around 17,000bp pooling PCR fragments and around 192 individual samples on a single Miseq run? Are there benefits of considering one over the other or protocols to follow for sample size and barcoding thanks for the help

05-22-2019, 01:47 PM	#2
SNPsaurus Registered Vendor Location: Eugene, OR Join Date: May 2013 Posts: 512	Can you multiplex that much on a Miseq and get enough reads per sample to reconstruct the mitogenome? I guess if the nuclear genome isn't large then a reasonable fraction of reads will go to the mitogenome. You can use partial reaction of just Nextera DNA Flex and get good results. That might be the cheapest route. __________________ Providing nextRAD genotyping and PacBio sequencing services. http://snpsaurus.com

05-23-2019, 12:11 PM	#4
SNPsaurus Registered Vendor Location: Eugene, OR Join Date: May 2013 Posts: 512	I see, that makes sense for amplified mtDNA. You can combine the Nextera Flex and Nextera XT barcode sets for Nextera Flex preps. We do that for increased multiplexing. __________________ Providing nextRAD genotyping and PacBio sequencing services. http://snpsaurus.com

05-23-2019, 06:02 AM	#3
frascom Junior Member Location: Kansas Join Date: May 2019 Posts: 4	I have amplified the mitogenome separately so there should no concern of reads. The main aim is trying to save some costs due to barcoding 192 individuals whilst achieving a high quality of reads. The benefit of NexteraXT I think is the availability of barcodes index for up to 384 samples. Although another option would be to use propriety barcodes so save a lot of money

05-23-2019, 08:25 PM	#5
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,230	plexWell 96 or 384 could fit your library prep. It seems to be cheap and also quick. https://seqwell.com/product/pw96/