DNA library preps

 

Prep type Recommended sample amount Min. conc. Min. volume Accepted buffers

Illumina® DNA Prep, Tagmentation*

(previously known as 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 ® Sonic +PCR**

100 - 500 ng

1 ng/μl**

100 μl

Low TE (10 mM Tris, 0.1 mM EDTA). 

Swift 2S ® Sonic, PCR-free** 500-1000 ng 10 ng/μl**

100 μl

Low TE (10 mM Tris, 0.1 mM EDTA). 

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 Illumina DNA prep (tagmentation) 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 submissions, all samples should be normalized before submitting. The more DNA you provide, the fewer PCR cycles are used in the prep. 

 

Library prep protocols:

Users may choose between library preparation by transposon tagmentation (Illumina® DNA Prep, Tagmentation) or by adapter ligation based preps with fragmentation by sonication (Swift 2S sonic DNA or SMARTer ThruPLEX® low-input).

Where DNA amounts allow  (≥ 200 ng for Swift 2S), 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. 

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 Nov. 15, 2021 2:39 PM