Nebnext ultra 2 dna library preparation kit

cfDNA was used for the preparation of the MeDIP-seq library with some modifications [27 (link)]. Briefly, we used the Illumina NEBNext Ultra II DNA Library Preparation Kit (NEB, E7645) and ligated ~ 50 ng of cfDNA to the Illumina adapter according to the manufacturer's instructions. The resulting library was denatured at 95 °C for 10 min, immediately incubated on ice for 10 min, and then immunoprecipitated with 5-methylcytosine (5-mC) monoclonal antibody (Epigentek, A-1014). The MeDIP DNA was amplified with Q5 high-fidelity DNA polymerase (NEB, M0491), and the amplified products were purified with AMPure XP beads (Beckman). The amplified libraries were evaluated using a Bioanalyzer 2100 system (Agilent Technologies), and deep sequencing was performed using an Illumina HiSeq 2000 system.

In situ Hi-C experiments were performed according to Concia et al.^{3 (link)} using DpnII enzyme (New England Biolabs). DNA libraries were prepared using NEBNext Ultra II DNA library preparation kit (NEB) according to the manufacturer’s instructions (10 cycles for the PCR amplification step). DNA libraries were checked for quality and quantified using a 2100 Bioanalyzer (Agilent) and the libraries were subjected to 2 × 75 bp high-throughput sequencing by NextSeq 500 (Illumina). Two independent biological replicates were generated.

Extracted genomic DNA was sheared to 100–400 bp (mean distribution 150 bp) using an LE220 ultrasonicator (Covaris). Libraries were prepared (NEBNext Ultra II DNA Library preparation kit, New England Biolabs) using initial adaptor ligation and barcoding with unique dual indexed barcodes (Integrated DNA Technologies). Dual indexed samples were amplified (6 cycles of PCR, KAPA HiFi kit, Roche), quantified (Accuclear dsDNA Quantitation Solution, Biotium), then pooled in pre-assigned groups of 48 or 32 to generate equimolar pools on the basis of total DNA concentration. Pooled DNA (500 ng) was hybridized using 120-mer RNA baits (SureSelect Target enrichment system, Agilent technologies; bait design ELID ID 0616571)^{4 (link),37 (link)}. Enriched libraries were sequenced on Illumina HiSeq 4000 to generate 150 bp paired-end reads at the Wellcome Sanger Institute (UK) as previously described³⁸ . For one rabbit-passaged sample from Melbourne, Australia (TPA_AUSMELT-1)³⁰ , genomic DNA extracted from historically archived tissue lysate was sequenced on Illumina NextSeq 500 (150 bp paired-end reads, Nextera DNA Flex libraries) without any previous enrichment to an estimated 1 Gb per sample at the Doherty Institute (Australia).

V. cholerae carrying a plasmid encoding tcaP was used in a plaque assay as described above. As a control, ICP1_2011_Dha_A was used to infect V. cholerae expressing an empty vector. Plaques that formed on the tcaP-expressing strain were picked and purified on that strain two more times by plaque assay. High-titer phage stocks were prepared by sodium chloride (1 mM) polyethylene glycol 8000 (10%) precipitation or by centrifugation (26,000 × g for 90 min) and stored in STE (5 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA). Prior to collecting genomic DNA (gDNA), phage stocks were treated with DNase for 30 min at 37°C to remove non-encapsidated DNA, then the enzyme was heat inactivated. gDNA was collected using a QIAGEN DNeasy blood and tissue DNA purification kit (QIAGEN, 69506) according to the manufacturer’s protocols and genomic libraries were prepared for Illumina sequencing using the NEBNext Ultra II DNA Library preparation kit (NEB #E7645, E7103) as described in the manufacturer’s protocols. Using an Illumina HiSeq4000 (University of California, Berkeley QB3 Core Facility), samples were sequenced by paired-end (2 × 150 bp). The genomes were assembled using SPAdes and analyzed using BreSeq (v0.33).

VZV genotyping and a search for co-infection was performed on a CSF sample from case 13, using mNGS [7 (link),8 (link)]. Total nucleic acid was extracted from 90 μL of CSF using the Zymo Quick-DNA/RNA MagBead (Zymo Cat. No. R2130) via the Agilent Bravo liquid handling robot (Santa Clara, CA, USA). The nucleic acid was then divided, with half undergoing DNAse treatment to isolate RNA for RNA sequencing (RNA-seq) and the remainder being used for DNA sequencing (DNA-Seq).
RNA-Seq libraries were prepared using the New England Biolabs’ NEBNext Ultra II RNA library preparation kit (NEB Cat No. E7770, Ipswich, MA, USA), DNA libraries were prepared using the New England Biolabs’ NEBNext^® Ultra™ II DNA Library preparation kit (NEB Cat No. E7645), both using the Echo Labcyte 525 and Agilent Bravo robots with a previously described protocol. Host ribosomal RNA depletion was performed using the Qiagen QIAseq FastSelect RNA removal kit (Qiagen Cat No. 333180, Hilden, Germany) at 1:100 dilution. Pooled libraries were size selected using Ampure beads. Final libraries were then sequenced on an Illumina Novaseq 6000 (San Diego, CA, USA) using 146 base pair paired-end sequencing. The mNGS workflow events are also illustrated in Figure 1.