Aug 24, 2020
nCoV-2019 McGill Nextera Flex LibPrep Protocol
Forked from a private protocol
- 1McGill University
- Coronavirus Method Development Community
- McGill Genome Centre
Protocol Citation: Marie-Michelle Simon, Sarah J Reiling, Ioannis Ragoussis 2020. nCoV-2019 McGill Nextera Flex LibPrep Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bjgnkjve
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 07, 2020
Last Modified: August 24, 2020
Protocol Integer ID: 40174
Abstract
How the Nextera DNA Flex Assay Works
The Nextera DNA Flex library prep kit uses a bead-based transposome complex to tagment genomic DNA, which is a process that fragments DNA and then tags the DNA with adapter sequences in one step. After it is saturated with input DNA, the bead-based transposome complex fragments a set number of DNA molecules. This fragmentation provides flexibility to use a wide DNA input range to generate normalized libraries of consistent tight fragment size distribution. Following tagmentation, a limited-cycle PCR adds Nextera DNA Flex-specific index adapter sequences to the ends of a DNA fragment. This step enables capability across all Illumina sequencing platforms. A subsequent Sample Purification Beads (SPB) cleanup step then purifies libraries for use on an Illumina sequencer. The double-stranded DNA library is denatured before hybridization of the biotin probe oligonucleotide pool.
PCR Amplicons for Nextera Flex
When starting with PCR amplicons, the PCR amplicon must be > 150 bp. The standard clean up protocol depletes libraries < 500 bp. Therefore, Illumina recommends that amplicons < 500 bp undergo a 1.8 x sample purification bead volume ratio to supernatant during Clean Up Libraries on page 11. Shorter amplicons can otherwise be lost during the library cleanup step. Tagmentation cannot add an adapter directly to the distal end of a fragment, so a drop in sequencing coverage of ~50 bp from each distal end is expected. To ensure sufficient coverage of the amplicon target region, design primers to extend beyond the target region by 50 bp per end.
Amplicon Quantification and normalisation
Amplicon Quantification and normalisation
Use the 30 ul with 150 ng DNA for Nextera Flex Tagmentation.
Protocol can be found here:
Start on page 7 with the Tagmentation.
Tagment Genomic DNA
Tagment Genomic DNA
Note
For automated protocol : at step 7, 22 ul tagmentation master mix is transfered.
Post Tagmentation Cleanup
Post Tagmentation Cleanup
Pool Libraries
Pool Libraries
Amplify Tagmented DNA
Amplify Tagmented DNA
Note
For automated protocol : at step 9, 44 ul tagmentation master mix is transfered.
Note
At step 14, beads can be resuspended in 62 uL RSB , followed by a 60 uL transfer of supernatant at step 18. This is used to decrease the concentration of the final pool in order to facilitate the QC step.
Check Library Quality
Check Library Quality
Dilute Libraries to the Starting Concentration
Dilute Libraries to the Starting Concentration