DNA library preps

 

Prep. type Min. amount Recommend amount Max. volume Accepted buffers
Nextera Flex prep* 2 ng* 200 - 1000 ng 60 μl

10 mM Tris

(TE also OK if conc > 40 ng/μl)

TruSeq Nano, 350 bp insert 0.2 μg 1 μg 130 μl 10 mM Tris, TE
TruSeq Nano, 550 bp insert 0.4 μg 2 μg 130 μl 10 mM Tris, TE
PCR-free prep 2 μg (measured by Qubit, NOT nanodrop) 10 μg (measured by Qubit, NOT nanodrop) 130 μl (min conc 30 ng/μl 10 mM Tris, TE

Input requirements for DNA library preps. *For Nextera submissions,samples on any one plate submitted should all fall within the following ranges of DNA amounts: 2-20 ng, 20-50 ng, 50-100 ng or 100-1000 ng (optimum) in a maximum volume of 60 µl. If sample concentrations span these ranges, samples of similar concentration should be grouped and submitted on separate plates. Concentrations must be determined by a fluorometric method such as Invitrogen´s Qubit, not spectrophotometric methods such as Nanodrop. It is not recommended to submit less than 100 ng DNA except in the case of small (e.g. bacterial) genomes.

Library prep protocols:

Users may choose between library preparation by transposon tagmentation (NexteraTM Flex) or by sonication and adapter ligation reagents (TruSeqTM Nano, TruSeqTM PCR-free, or SMARTer ThruPLEX® low-input). Our favoured default procedure employs the NexteraTM Flex reagents, entailing 6 cycles PCR, which allows a rapid turnaround and is relatively insensitive to DNA input amounts (see the table above). Where DNA amounts allow (≥2 µg), PCR-free preps are recommended when the organism to be sequenced has high or low GC content. When limiting sample amounts are available (100 pg – 50 ng), SMARTer ThruPLEX® preps should be selected. TruSeqTM Nano reagent is a legacy product that in most cases has been superseded by the NexteraTM Flex reagent, but is still available.

Note that PCR-free libraries, due to being partially single stranded, do not store well. PCR-free libraries over 2 months of age must be re-QC’d before sequencing, and may not retain sufficient activity to provide data. PCR-free libraries older than 6 months are unlikely to be sequence-able.

DNA for sequencing should be treated with RNase before final cleanup and submission.

Library preps from WGA material

Whole-genome amplified (WGA) and whole-transcriptome amplified (WTA) samples are frequently problematic, so will only be sequenced at the risk of the submitter.

Published Mar. 20, 2020 11:09 AM - Last modified Mar. 30, 2020 2:10 PM