TITLE: Library Construction Protocols
AUTHORS: Graham J Etherington, Darren Heavens, David Baker, Ashleigh Lister, Rose McNelly, Gonzalo Garcia, Bernardo Clavijo, Iain Macaulay, Wilfried Haerty, Federica Di Palma

[DESCRIPTION]
Amplification Free Paired End Library Construction Protocol.

[STEPS]
SECTION: PCR-free Illumina 2500
1. A total of 600 ng of DNA was sheared in a 60 µL volume on a Covaris S2 (Covaris, Massachusetts, USA) for 1 cycle of 40 s with a duty cycle of 5%, cycles per burst of 200 and intensity of 3.
SECTION: PCR-free Illumina 2500
2. The fragmented molecules were then end repaired in 100 µL volume using the NEB End Repair Module (NEB, Hitchin, UK) incubating the reaction at 22 °C for 30 min.

SECTION: PCR-free Illumina 2500
3. Post incubation 58 µL 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.

SECTION: PCR-free Illumina 2500
4. This is then washed twice with 70% ethanol and the end repaired molecules eluted in 25 µLNuclease free water (Qiagen, Manchester, UK).

SECTION: PCR-free Illumina 2500
5. End repaired molecules were then A tailed in 30 µL volume using in the NEB A tailing module (NEB) incubating the reaction at 37 °C for 30 min.

SECTION: PCR-free Illumina 2500
6. To the A tailed library molecules 1 µL of an appropriate Illumina TruSeq Index adapter (Illumina, San Diego, USA) is added and mixed, then 31 µL of Blunt/ TA ligase (NEB) is added and incubated at 22 °C for 10 min.

SECTION: PCR-free Illumina 2500
7. Post incubation 5 µL of stop ligation is added and the reaction incubated at Room temperature for 5 min.

SECTION: PCR-free Illumina 2500
8. Following this incubation 67 µL beads of CleanPCR beads (GC Biotech, Alphen aan den Rijn, The Netherlands) were added and the DNA precipitated onto the beads.

SECTION: PCR-free Illumina 2500
9. The samples are then washed twice with 70% ethanol and the end repaired molecules eluted in 100 µL nuclease free water.

SECTION: PCR-free Illumina 2500
10. 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 25 µL Resuspension Buffer (Illumina).

SECTION: PCR-free Illumina 2500
11. Library QC was performed by running a 1 µL aliquot on a High Sensitivity BioAnalyser chip (Agilent, Stockport, UK) and the DNA concentration measured using the High Sensitivity Qubit (Thermo Fisher, Cambridge, UK).

SECTION: PCR-free Illumina 2500
12. 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.
SECTION: Illumina NovaSeq 6000
13. 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).
SECTION: Illumina NovaSeq 6000
14. 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.
SECTION: Illumina NovaSeq 6000
15. 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.