DNA library preps

 

Prep type Recommended sample amount Min. conc. Min. volume Accepted buffers
Illumina Nextera™ DNA Flex prep* 200 - 1000 ng  3 ng/μl 60 μl

10 mM Tris

(TE also OK if conc > 40 ng/μl)

Swift 2S ® Turbo, 350/550 bp insert, +PCR**

100 - 500 ng

3 ng/μl** 40 μl up to 1 mM EDTA (optimal 0.1 mM)
Swift 2S ® Turbo, 350 bp insert, PCR-free** 500 ng 15 ng/μl** 60 μl up to 1 mM EDTA (optimal 0.1 mM)
TruSeq™ Nano, 350 bp insert 1 μg 5 ng/μl 110 μl 10 mM Tris, TE
TruSeq™ Nano, 550 bp insert 2 μg 10 ng/μl 110 μl 10 mM Tris, TE
TruSeq™ PCR-free prep 5 μg (measured by Qubit) 40 ng/μl 110 μl 10 mM Tris, TE

Input requirements for DNA library preps. 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. If your samples do not meet the requirements above, contact us and we will try to find a solution.

*For Nextera Flex 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. 

**For Swift 2S subissions, all samples should be normalized to 3-5 ng/μl (PCR-prep) or 15 ng/ μl (PCR-free prep) before submitting. The more DNA you provide, the fewer PCR cycles are used in the prep. This prep is only available for submission of minimum of 48 samples. 

 

Library prep protocols:

Users may choose between library preparation by transposon tagmentation (Nextera™ Flex) or by adapter ligation based preps, with either ezymatic fragmentation (Swift 2S® Turbo) or fragmentation by sonication (TruSeq™ Nano, TruSeq™ PCR-free, or SMARTer ThruPLEX® low-input).

Where DNA amounts allow  (≥2 µg for Truseq prep, and > 200 ng for Swift 2S Turbo), 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. TruSeq™ Nano reagent is a legacy product that in most cases has been superseded by the Nextera™ Flex reagent and Swift 2S Turbo reagent, but is still available.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 Oct. 2, 2020 2:36 PM