Miseq sequencing machine

High-throughput sequencing of the parotid gland sample from the s.c. inoculated guinea pigs at 6 dpi was performed on an Illumina MiSeq sequencing machine. The genome sequences of ZIKV were assembled by mapping the reads to the reference genome of ZIKV GZ01. The mapping and iSNV detection were processed with CLC Genomic Workbench. The site with substitution frequency above 5% was considered as an iSNV^{22 (link)}.
For determination of consensus sequence containing the M469V substitution, RT-PCR were performed with RNAs extracted from various tissue samples from the ZIKV-infected guinea pigs. The primers used for RT-PCR and Sanger sequencing include: ZIKV-F3 (1420–1440): 5′-GGAAGCCTAGGACTTGATTGT-3′; ZIKV-R3(2376-2397): 5′-CGAGCACCCCACATCAGCAGAG-3′. Sequence fragments were assembled into a consensus sequence with DNA STAR software, version 7.0.

Using approximately 50 ng of the extracted genomic DNA, we performed TruSeq Custom Amplicon v2.0 (Illumina) targeted re-sequencing experiments on a selected set of pancreatic cancer driver genes using a custom pancreatic cancer panel. The custom pancreatic cancer panel was established using Illumina TruSeq Amplicon-Cancer Panel platform which provided custom-designed, optimized oligonucleotide probes for sequencing mutational hotspots of pancreatic cancer in >117 kilobases (kb) of target genomic sequence. Within this highly multiplexed, single-tube reaction, 20 genes are targeted with 1242 Amplicons. Each amplicon had one pair of oligos designed to hybridize to the region of interest. The reaction was then followed by extension and ligation to form DNA templates consisting of regions of interest flanked by universal primer sequences. These DNA templates were amplified by indexed primers and then pooled into a single tube in order to sequence on an Illumina MiSeq sequencing machine. Canonical variant alleles for KRAS and TP53 were preselected from TCGA and ICGC mutation databases as the most frequently recurrent hotspot variant alleles in PDAC.

The BG01-4^TM and BG01-WT isolates were streaked and grown on blood agar (Table S2) for 48 h at 37 °C. DNA was then extracted from BG01-4^TM and BG01-WT, and then prepared with the Zymo Research fungal/bacterial DNA miniprep kit (Supplementary Information 1). The DNA was then further prepared using a QIAseq FX DNA Library Kit [24 (link)] (Supplementary Information 2) for next generation sequencing (NGS) on an Illumina Miseq sequencing machine.

MV RNA was isolated from UPEC 536. Libraries were prepared using a TruSeq small RNA library kit (Illumina), retaining the fractions from the gel isolation step that correspond to 15-50bp and 50-200bp. These were run on an Illumina MiSeq sequencing machine (2x50bp reads and 2x250bp reads, respectively), one library per MiSeq lane.
Libraries of longer RNAs were prepared using an Illumina TruSeq stranded mRNA kit, excluding the poly-A selection step and without ribo-zero depletion (no removal of ribosomal RNAs). The ribo-zero step was deliberately excluded in order to provide us with a full understanding of the RNA content of UPEC MVs, including these highly abundant ribosomal RNAs. 60ng of total RNA was used as an input for the fragment, prime and finish step of the protocol. One library was run on each lane of a HiSeq 2500 sequencing machine (2x100bp). RNA-sequencing was performed as a contract service by New Zealand Genomics Ltd.

Genomic DNA was purified from the EAEC isolates using the DNeasy Blood and Tissue kit (Qiagen). Sequencing was carried out with the Illumina technology on a MiSeq sequencing machine, using the Nextera XT Library preparation protocol (Illumina) for obtaining paired-end reads of 150 bp or 250 bp. Raw data for all isolates were deposited at the European Nucleotide Archive (ENA).
WGS data were pre-processed employing a QC-pipeline (available at https://github.com/ssi-dk/SerumQC). Sequences were removed in the case of contamination with more than 5% of another genera and sequences representing isolates with genome sizes outside the range of 4.64 Mbp-5.56 Mbp. Sequences were removed from the dataset if assemblies comprised of > 350 contigs. De novo assembly was carried out using CLC Genomics Workbench 10.

DNA was extracted using the Qiagen DNeasy blood and tissue kit for skin mucosa, and TRIzol organic phase followed by BEB (back extraction buffer) and PCI (phenol/chloroform/isoamyl alcohol 25:24:1) solutions for all gut samples. The V3–V4 hypervariable region of the universal rDNA 16S gene (Werner et al. 2012) was amplified using universal specific primers. The libraries of amplicons were prepared using a set of 384 combinations of adaptors, processed in one sequencing run, on an Illumina Miseq sequencing machine. Reactions of PCR were verified by electrophoresis on 2% agarose gel, purified, and quantified by fluorescence for the double-strand DNA concentration using Quant-iT™ PicoGreen™ dsDNA Assay Kit (Thermo Fischer Scientific).

Viral RNA was isolated using a PureLink RNA minikit (Life Technology, USA) according to the manufacturer's instructions. The genome cDNA was obtained by reverse transcription (RT) using SuperScript III (Life Technology, USA). For determination of consensus sequence, PCR products were directly sequenced by Sanger sequencing in both directions using virus-specific primers (Table S1). Sequence fragments were assembled into a consensus sequence with DNASTAR software, version 7.0.
High throughput sequencing was performed on an Illumina MiSeq sequencing machine. The genome sequences of the viruses were assembled by mapping the reads to the reference genome of ZIKV strain GZ01/2016. Genomic mapping and single nucleotide polymorphism (SNP) detection were processed with CLC Genomic Workbench. The site with substitution frequency above 5% was considered as a single nucleotide variant (iSNV).