Editing NGS Library Preparation (section)

=== Library Preparation for Sequencing ===

==== Norovirus amplicon preparation for Sanger sequencing. ====

Reverse transcription and first strand cDNA synthesis were performed using the Accuscript High Fidelity 1st strand cDNA synthesis kit (Agilent, Santa Clara, California, USA) following the manufacturer's published protocol. PCR amplification was then performed using specific in-house primers covering the seven overlapping amplicons of the genome of Norovirus GII.4 (see [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066129#pone.0066129.s001 Table S1]), including internal primers to increase depth of coverage using Sanger sequencing. PCR products were then confirmed by agarose gel electrophoresis. The reaction products were separated and detected with a Prism 3730 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA). Sequences were assembled from the resultant chromatograms with the STADEN suite of computer programs [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066129#pone.0066129-Staden1 [23]]. Primer sequences at the 5′ and 3′ termini of the overlapping PCR amplicons were excluded.

==== Hepatitis C Sanger sequencing. ====

One-step reverse transcription and first-round PCR in two reactions amplified a 4-kb and a 7-kb product. Second-round nested PCR reactions used >20 PCR primers in pairs generating 10 overlapping viral genomic fragments each of approximately 1 kb, that were sequenced and aligned manually to a reference sequence as previously described [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066129#pone.0066129-Humphreys1 [24]].

==== Illumina RNA-Seq Library Preparation. ====

Total RNA quantity and integrity were assessed using Quant-IT RiboGreen RNA Assay Kit (Invitrogen, Carlsbad, CA, USA) and Agilent Tapestation 2200 R6K. Libraries for Illumina sequencing were constructed from 100 ng of total RNA using the NEBNext mRNA Sample Prep Kit 1 (New England Biolabs, Ipswich, MA, USA), following the manufacturers' guidelines with minor modifications: end repair in 50 µl reaction volume (40 µl DNA, 5 µl buffer and 5 µl enzyme), post fragmentation clean-up with 1:2.8× volume Agencourt Ampure RNAClean XP (Beckman Coulter, Pasadena, CA, USA); post cDNA synthesis clean-up with 1:1.25× volume Ampure XP Beads; post end repair, A-Tailing and adapter ligation clean-ups with 1:1.8× volume Ampure XP Beads and post-PCR library clean-ups with 1:1× volume Ampure XP Beads. Additionally, upon ligation of Illumina Adapters (Multiplexing Sample Preparation Oligonucleotide Kit) each library was size selected with two Ampure Bead steps (firstly, 1:0.7× volume and secondly, the supernatant from the first bind was taken for a 1:1.7× volume clean-up), selecting 200–600 bp fragments in 30 µl 10 mM Tris-Cl, pH 8.5. Pre-PCR workflow was partially performed using a Beckman Biomek FX and post-PCR steps were performed using a Beckman Biomek NX<sup>P</sup> and Biomek 3000.



The following custom primers (25 µM each) were used for the PCR enrichment step:



Multiplex PCR primer 1.0



<span class="gene">5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′</span>



Index primer



<span class="gene">5′-CAAGCAGAAGACGGCATACGAGAT</span>[INDEX]<span class="gene">CAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′</span>



Amplified libraries were analysed for size distribution using the Agilent Tapestation 2200 D1K. Libraries were quantified by quantitative RT-PCR using Agilent qPCR Library Quantification Kit and a Mx3005P instrument (Agilent) and relative volumes were pooled accordingly. Finally, a second quantitative RT-PCR was performed to measure the relative concentration of the pool compared to a previously sequenced mRNA library in order to determine the volume to use for sequencing.

==== Improved Fragmentation Library Preparation for Illumina. ====

To increase the library insert size of the sequenced libraries and to further investigate secondary structures within the Norovirus genome, a modified fragmentation method was developed. The total RNA was fragmented using 2 µl fragmentation buffer diluted 1:4 in nuclease-free water (fragmentation buffer included in the NEBNext mRNA Library Prep Master Mix Set), incubated at 94°C for 5 minutes, placed immediately to ice and followed by adding 2 µl stop solution (not diluted). Post-fragmentation clean-up through to adapter ligation clean-up were as above. Size selection was omitted to avoid losing longer fragments, and instead, an additional bead clean-up after ligation was performed to remove any remaining adapter dimer prior to PCR amplification.

==== Illumina Amplicon Library Preparation. ====

Amplicons were quantified and quality assessed using Quant-IT Qubit dsDNA High Sensitivity Assay (Invitrogen) and 1% E-gel (Invitrogen) respectively. Fifty nanograms were sheared to 400 bp using Covaris™ DNA Shearing (Woburn, MA, USA) and concentrated using 1:1× volume Ampure XP Beads. Eluted DNA fragments were then processed through standard library preparation procedures (end repair through to Ampure Bead size selection using the NEBNext DNA Library Prep Master Mix Set for Illumina). Post-reaction clean-ups were 1:1.8× volume Ampure XP Beads and post-PCR libraries were cleaned with 1:1× volume Ampure XP. Ten cycles of PCR amplification were performed using custom primers as above, and the final library was pooled with Norovirus positive samples from the pilot study.

==== Sequencing. ====

Multiplex libraries were prepared using barcoded primers and a median insert size of 150 bp (increased to 200 bp with the modified method). The pooled libraries from 3 initial Norovirus and 2 HCV samples were sequenced on an Illumina MiSeq with 150 bp paired end reads following standard Illumina protocols. An average of 0.5 Gb of sequence was produced per sample. A larger pool of 77 Norovirus samples was sequenced on an Illumina HiSeq 2000 with 100 bp paired end reads following standard Illumina protocols.

==== Data analysis. ====

Reference sequences for each organism were obtained from GenBank. For Norovirus the sequences were GII.4, and the Norovirus Hu/GII.4/Orange/NSW001P/2008/AU was used as a reference (accession number GQ845367). This was selected as the most common match to the Sanger sequenced 288 bp RT-PCR amplicon used in the diagnostic RT-PCR stage. For HCV the reference sequence used was the subtype 3a strain (accession number AF046866).



Sequences were mapped to the organism-specific reference using Stampy v1.0.14 [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066129#pone.0066129-Lunter1 [25]] with no BWA pre-mapping. Bases and single nucleotide variants (SNVs) were called using the SAMtools “mpileup” command with options ‘-E -M0 -Q30 -q30 -o40 -e20 -h100 -m2 -D -S’ and BCFtools [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066129#pone.0066129-Li1 [26]]. Sites were filtered to avoid unreliable calls using the following criteria:

#A minimum depth of 5 reads at each position
#A minimum average base quality of 10
#A minimum SNV quality of 25
#At least 75% of reads at the position support the call and the position was called as homozygous



Positions were called as heterozygous based on the SAMtools genotype calls. An evolutionary tree was created using BEAST (Bayesian evolutionary analysis sampling trees) depicting all the full genomic sequences with relatedness. Analysis was performed using BEAST v.1.7.5 combining two random number seed chains (10 million iterations each, saving 1 in 1,000 iterations, with a 1 million iteration burn-in) using: HKY substitution; estimated frequency; strict clock; and constant population size coalescent tree prior. This maximum clade credibility tree was computed using TreeAnnotator v.1.7.5 and plotted with Figtree v.1.4.0. [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066129#pone.0066129-Drummond1 [27]].



The sequences reported in this paper have been deposited in the European Nucleotide Archive Sequence Read Archive under study accession number ERP002219. The Sanger sequences used for validation for Norovirus comparison have been submitted to EMBL under accession numbers HF952119-HF952135. For HCV, the Sanger sequences used for comparison were Genbank accession numbers GQ356201 and KC836883.
[[Category:Protocols]]