For genomic DNA extraction, ∼ 45 ml of each AOM enrichment (E20, G37, G60) were harvested by centrifugation (5000 × g for 15 min). DNA was extracted from the pellet according to Zhou and colleagues (1996) following the protocol described by Wegener and colleagues (2016 ). For metagenome sequencing, 2–4 µg of high‐molecular‐weight DNA was used for PCR‐free TruSeq paired‐end and mate‐pair library preparation, following the instructions of the respective Illumina library preparation kits. Paired‐end libraries were prepared from ∼ 500 bp DNA fragments and mate‐pair libraries from DNA fragments of ∼ 5000 bp. Libraries were sequenced on an Illumina MiSeq platform using sequencing chemistry to generate 250 bp reads.
Library preparation kits
Manufactured by Illumina
Library preparation kits are a set of reagents and consumables used to prepare DNA or RNA samples for sequencing on Illumina platforms. The core function of these kits is to convert the original nucleic acid samples into sequencing-ready libraries that can be loaded onto Illumina sequencing instruments.
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5 protocols using library preparation kits
1
Genomic DNA Extraction and Metagenome Sequencing
2
Illumina Paired-end Sequencing of Plant Genomes
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For the 130 samples, genomic DNA was extracted from leaf samples with the Qiagen DNeasy plant kit. The sequencing libraries were constructed with an insert size of ~ 300 bp using Illumina library preparation kits and were sequenced using the Illumina HiSeq 2500 platform with 2 × 150 bp paired reads to a target coverage of 25× following ref. [107 (link)]. The raw sequencing data has been deposited in the Short Read Archive at NCBI under BioProject ID: PRJNA731597.
3
Viral Genome Sequencing and Variant Analysis
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The ancestor virus population and virus populations for each line at the four assayed time points (37 samples) were next prepared for sequencing. First, extracted occlusion bodies (OBs) were rinsed in 0.1% SDS and purified in a Percoll gradient as in (Gilbert et al., 2014) (link). OBs were then dissolved in 0.5M Na 2 CO 3 and DNA was extracted with a QIAamp DNA kit. Library preparation and sequencing was conducted at the UC Berkeley QB3 center on non-amplified DNA.
150bp paired end libraries were generated with Kapa Biosystems library preparation kits and multiplexed to run on one lane of an Illumina MiSeq platform. Reads were then de-multiplexed and aligned to the PiGV reference genome [GenBank: KX151395] using bowtie2 (Harrison et al., 2016; (link)Langmead and Salzberg, 2012) (link). The resulting alignments for each sample had 99.99-100% genome coverage, 51-100 mean coverage depth, and 40.2-41 mean MapQ scores. Variant calls were made using VarScan (Koboldt et al., 2012) (link) in Galaxy, calling indels and SNPs separately using a minimum coverage of 20 and a minimum alternate allele read count of 2. The Galaxy history can be viewed here: https://usegalaxy.org/u/evisher/h/specialization-sequence-variants.
150bp paired end libraries were generated with Kapa Biosystems library preparation kits and multiplexed to run on one lane of an Illumina MiSeq platform. Reads were then de-multiplexed and aligned to the PiGV reference genome [GenBank: KX151395] using bowtie2 (Harrison et al., 2016; (link)Langmead and Salzberg, 2012) (link). The resulting alignments for each sample had 99.99-100% genome coverage, 51-100 mean coverage depth, and 40.2-41 mean MapQ scores. Variant calls were made using VarScan (Koboldt et al., 2012) (link) in Galaxy, calling indels and SNPs separately using a minimum coverage of 20 and a minimum alternate allele read count of 2. The Galaxy history can be viewed here: https://usegalaxy.org/u/evisher/h/specialization-sequence-variants.
4
SARS-CoV-2 Whole Genome Sequencing Workflow
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Whole genome sequencing (WGS) was performed on all RT-PCR positive samples [9 (link)]. Viral amplicons were sequenced using Illumina library preparation kits (Nextera) and sequenced on Illumina short-read sequencing machines (Nextseq or Hiseq). The bioinformatics protocol to generate consensus sequences utilised Trimmomatic, BWA (mapping), and an in-house variant caller (quasibam) to align against a SARS-CoV-2 reference genome (NC_045512.2). Consensus sequences were generated using a depth cut-off of 20 reads and ambiguities called where a minority variant detected at ≥20%, these were aligned using MAFFT (Multiple Alignment using Fast Fourier Transform, version 7.310), manually curated and maximum likelihood phylogenetic trees derived using IQtree (version 2.04). Genomes were included in analysis where the coverage of the reference genomes was ≥80%. Completed viral genomes were deposited in GISAID (Supplementary Table 1) [10 (link)].
5
SARS-CoV-2 Genomic Surveillance Protocol
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Nose and throat swabs from residents and staff were sent by courier to the UKHSA Colindale Reference Laboratory for RT-PCR testing using a SARS-CoV-2 assay with E and Orf1ab gene targets as previously described (5 (link)). Some PCR testing of staff was also performed via community testing programmes in government Lighthouse laboratories. UKHSA samples with a cycle threshold (Ct) value less than 35 underwent whole genome sequencing. Viral amplicons were sequenced using Illumina library preparation kits (Nextera) and sequenced on Illumina shortread sequencing machines (Nextseq or Hiseq). The bioinformatics protocol to generate consensus sequences utilised Trimmomatic, BWA (mapping), and an in-house variant caller (quasibam) to align against a SARS-CoV-2 reference genome (NC_045512.2). Consensus sequences were generated using a depth cut-off of 20 reads. Genome lineages were allocated where the coverage of the reference genomes was 80% or more. Serological testing was performed using Roche Elecsys® Anti-SARS-CoV-2 S and N antibody testing according to manufacturers instructions.
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