NGS library prep method for RRS/GBS/RADseq of degraded/ancient DNA?

BioGenomics · 01-12-2015, 08:51 PM

Hi,

I am looking for the best NGS library prep method for RRS/GBS/RAD sequencing of degraded/low/template/ancient DNA ? Any good protocol available to genotype 5-10K SNPs in 100-200 inds reliably starting from already fragmented DNA ? Otherwise best SNP genotyping assay for such low qual DNA samples ?

Thanks

BG

nucacidhunter · 01-13-2015, 03:04 PM

Considering input DNA quantity and quality there are two options: 1- Illumina’s genotyping bead arrays. 2- For sequencing based approach preparing Illumina libraries using a low input kit followed by whole genome sequencing or a targeted capture approach (to reduce sequencing requirement). With current kits it is possible to perform capture with very low inputs (pg amount).

BioGenomics · 01-13-2015, 06:57 PM

Thanks nucacidhunter. Well as I don't have an extensive list of SNPs for my species (some RAD data, but no good flanking regions... I might try a capture method as there is a draft genome (set of contigs/scaffold). What techniques would be cost-efficient ? I heard MyBaits would be good ? Thanks again

nucacidhunter · 01-13-2015, 07:41 PM

MyBaits are definitely cost effective and there are some published papers using their baits. For library prep I think ThruPlex DNA-seq could be good option if you have limited material. You may adapt the approach that has been described here: http://rubicongenomics.com/products/...x-dna-seq-kit/ to MyBaits capture protocol.

BioGenomics · 01-13-2015, 09:59 PM

Looks promising indeed, how does this lib prep compare to the NEB Ultra DNA kit for instance ? Did you use it already ? Pricing ?

nucacidhunter · 01-14-2015, 12:38 AM

Quote:

Originally Posted by BioGenomics

Looks promising indeed, how does this lib prep compare to the NEB Ultra DNA kit for instance ? Did you use it already ? Pricing ?

I have not used it, but reviewing manufacturer data, related patents and published literature indicates higher performance for this kit as claimed. Pricing is depend on kit size, as always with larger kits prices gets lower and it is pretty close to NEB kits price. I assume prices would be different depending on location.

nucacidhunter · 01-14-2015, 09:46 PM

This paper also seems interesting for your application: http://journals.plos.org/plosone/art...l.pone.0096793

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01-12-2015, 08:51 PM	#1
BioGenomics Member Location: Belgium Join Date: Apr 2009 Posts: 24	NGS library prep method for RRS/GBS/RADseq of degraded/ancient DNA? Hi, I am looking for the best NGS library prep method for RRS/GBS/RAD sequencing of degraded/low/template/ancient DNA ? Any good protocol available to genotype 5-10K SNPs in 100-200 inds reliably starting from already fragmented DNA ? Otherwise best SNP genotyping assay for such low qual DNA samples ? Thanks BG

01-13-2015, 03:04 PM	#2
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	Considering input DNA quantity and quality there are two options: 1- Illumina’s genotyping bead arrays. 2- For sequencing based approach preparing Illumina libraries using a low input kit followed by whole genome sequencing or a targeted capture approach (to reduce sequencing requirement). With current kits it is possible to perform capture with very low inputs (pg amount).

01-13-2015, 06:57 PM	#3
BioGenomics Member Location: Belgium Join Date: Apr 2009 Posts: 24	Thanks nucacidhunter. Well as I don't have an extensive list of SNPs for my species (some RAD data, but no good flanking regions... I might try a capture method as there is a draft genome (set of contigs/scaffold). What techniques would be cost-efficient ? I heard MyBaits would be good ? Thanks again

01-13-2015, 07:41 PM	#4
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	MyBaits are definitely cost effective and there are some published papers using their baits. For library prep I think ThruPlex DNA-seq could be good option if you have limited material. You may adapt the approach that has been described here: http://rubicongenomics.com/products/...x-dna-seq-kit/ to MyBaits capture protocol.

01-13-2015, 09:59 PM	#5
BioGenomics Member Location: Belgium Join Date: Apr 2009 Posts: 24	Looks promising indeed, how does this lib prep compare to the NEB Ultra DNA kit for instance ? Did you use it already ? Pricing ?

01-14-2015, 09:46 PM	#7
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	This paper also seems interesting for your application: http://journals.plos.org/plosone/art...l.pone.0096793