Library Construction Protocols

A total of 600ng of DNA was sheared in a 60µL volume on a Covaris S2 (Covaris, Massachusetts, USA) for 1 cycle of 0h 0m 40s with a duty cycle of 5%, cycles per burst of 200 and intensity of 3.

The fragmented molecules were then end repaired in 100µL volume using the NEB End Repair Module (NEB, Hitchin, UK) incubating the reaction at 22°C for 0h 30m 0s.

Post incubation 58µL beads of CleanPCR beads (GC Biotech, Alphen aan den Rijn, The Netherlands) were added using a positive displacement pipette to ensure accuracy and the DNA precipitated onto the beads.

This is then washed twice with 70% ethanol and the end repaired molecules eluted in 25µLNuclease free water (Qiagen, Manchester, UK).

End repaired molecules were then A tailed in 30µL volume using in the NEB A tailing module (NEB) incubating the reaction at 37°C for 0h 30m 0s.

To the A tailed library molecules 1µL of an appropriate Illumina TruSeq Index adapter (Illumina, San Diego, USA) is added and mixed, then 31µL of Blunt/ TA ligase (NEB) is added and incubated at 22°C for 0h 10m 0s.

Post incubation 5µL of stop ligation is added and the reaction incubated at Room temperature for 0h 5m 0s.

Following this incubation 67µL beads of CleanPCR beads (GC Biotech, Alphen aan den Rijn, The Netherlands) were added and the DNA precipitated onto the beads.

The samples are then washed twice with 70% ethanol and the end repaired molecules eluted in 100µL nuclease free water.

Two further CleanPCR bead based purifications were undertaken to remove any adapter dimer molecules that may have formed during the adapter ligation step. The first with 0.9x volume beads, the second with 0.6x and the final library eluted in 25µL Resuspension Buffer (Illumina).

Library QC was performed by running a 1µL aliquot on a High Sensitivity BioAnalyser chip (Agilent, Stockport, UK) and the DNA concentration measured using the High Sensitivity Qubit (Thermo Fisher, Cambridge, UK).

To determine the number of viable library molecules the library was subjected to quantification by the Kappa qPCR Illumina quantification kit (Kapa Biosystems, London, UK) and a test lane run at 10pM on a MiSeq (Illumina) with 2x300bp reads to allow the library to be characterised prior to generation of the 60x coverage required on the Hiseq2500s (Illumina) with a 2x250bp read metric.

The libraries for this project were constructed at the Earlham Institute, Norwich, UK using

the KAPA High Throughout Library Prep Kit (Roche Part No: KK8234/07961901001) on the

Perkin Elmer Sciclone NGS Workstation liquid handling platform.

1μg of genomic DNA was sheared to 350bp using the Covaris LE220 Sonicator (Covaris and

Life Technologies), the ends of the DNA were repaired; 3' to 5' exonuclease activity removed

the 3' overhangs and the polymerase activity filled in the 5' overhangs creating blunt ends. A

single ‘A’ nucleotide was added to the 3’ ends of the blunt fragments to allow for the

ligation of barcoded adapters (12bp - Perkin Elmer NEXTFLEX-HT (NOVA-51474/5/6/7)) at a

concentration of 6μM prior to a double sided clean up using Beckman Coulter AMPure XP

beads (A63882). Adaptor ligated DNA was then enriched with 6 cycles of PCR (45 secs at

98°C, 6 cycles of: 15 secs at 98°C _30 secs at 60°C _30 secs at 72°C, 60 secs at 72°C, final

hold at 4°C).

The resulting libraries were QC’d using the Perkin Elmer GX Touch DNA High Sensitivity

assay (DNA High Sensitivity Reagent Kit CLS760672) and the concentrations determined with

a high sensitivity plate reader Quant-iT™ dsDNA Assay Kit, (ThermoFisher Q-33120). The

resulting libraries were then equimolarly pooled and q-PCR was performed on the pool prior

to sequencing using the KAPA qPCR kit which quantifies full-length library fragments by

using primers that anneal to the p5 and p7 sequences.

The library pool was diluted down to 0.65 nM using EB (10mM Tris pH8.0) in a volume of

18ul before spiking in 1% Illumina phiX Control v3 (Illumina, FC-110-3001). This was

denatured by adding 4ul 0.2N NaOH and incubating at room temperature for 8 mins, after

which it was neutralised by adding 5ul 400mM tris pH 8.0. The ExAmp master mix was

prepared by combining EPX1, EPX2, and EPX3 from the NovaSeq Xp 2-lane kit v1.0 as per the

manufacturer’s instructions before loading onto a NovaSeq SP flow cell which was loaded

onto the NovaSeq 6000 along with a NovaSeq 6000 SP cluster cartridge, buffer cartridge,

and 300 cycle SBS cartridge (Illumina, 20027465). The NovaSeq had NVCS v1.6.0 and RTA

v3.4.4 and was set up to sequence 150bp PE reads. The data was demultiplexed and

converted to fastq using bcl2fastq2.