TITLE: nCoV-2019 McGill Nextera Flex LibPrep Protocol
AUTHORS: Marie-Michelle Simon, Sarah Reiling

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">How the Nextera DNA Flex Assay Works </span></div><div class = "text-block">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. </div><div class = "text-block"><span style = "font-weight:bold;">PCR Amplicons for Nextera Flex</span></div><div class = "text-block"><span>When starting with PCR amplicons, the PCR amplicon must be > 150 bp. The standard clean up protocol depletes libraries < 500 bp. </span><span style = "font-weight:bold;">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. </span><span>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. </span></div><div class = "text-block"><span>More information can be found here: </span><a href="https://emea.support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/samplepreps_nextera/nextera_dna_flex/nextera-dna-flex-library-prep-reference-guide-1000000025416-07.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://emea.support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/samplepreps_nextera/nextera_dna_flex/nextera-dna-flex-library-prep-reference-guide-1000000025416-07.pdf</span></a></div></div>

[STEPS]
?. [Amplicon Quantification and normalisation]
Use the 30 ul with 150 ng DNA for Nextera Flex Tagmentation. Protocol can be found here:https://emea.support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/samplepreps_nextera/nextera_dna_flex/nextera-dna-flex-library-prep-reference-guide-1000000025416-07.pdfStart on page 7 with the Tagmentation.
?. [Tagment Genomic DNA]
For automated protocol : at step 7, 22 ul tagmentation master mix is transfered.
?. [Post Tagmentation Cleanup]
?. [Pool Libraries]
?. [Amplify Tagmented DNA]
For automated protocol : at step 9,  44 ul tagmentation master mix is transfered.
?. 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]
?. [Dilute Libraries to the Starting Concentration]