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155 protocols using kapa hyperplus kit

1

DNA Fragmentation and Library Preparation

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Genomic DNA (1 μg) was enzymatically fragmented to an average length of approximately 500–600 bp using the KAPA Hyper Plus kit (Roche, Cape Town, South Africa), following end repair and addition of adenosine at the 3′-ends of fragmented DNA. For sample traceability and library amplification, 5 μL each of 24 barcoded SeqCap index adaptors (kit A and kit B, 12 index adaptors per kit) (Roche Sequencing Solutions, Inc., Pleasanton, CA, USA) was ligated to DNA fragments of each individual sample using the KAPA Hyper Plus kit according to the manufacturer’s instructions. Adaptor-tagged library fragments were purified using Agencourt AMPure XP beads (Beckman Coulter, Inc., Brea, CA, USA) on the NGS Magna Stand Ch YS-Model (Nippon Genetics Co., Ltd.) according to the manufacturer’s instructions. In order to remove unwanted fragment sizes and low molecular weight material, such as adaptor dimers, that interfere with downstream library preparation steps (including cluster formation, sequencing, and analysis), double-sided size selection to keep fragments from 250 bp to 450 bp was performed using Agencourt AMPure XP beads before the adaptor-tagged purified libraries were subjected to PCR amplification.
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2

Plasmid Curing in E. coli and P. putida

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Two parallel cultures were initiated from each of three individual colonies of E. coli carrying pFREE and P. putida carrying pFREE-RK2. One culture was induced to activate the curing process while the other functioned as a control without induction of pFREE. Both cultures were grown at 30 °C shaking (250 rpm) for 24 h and genomic DNA was purified using the QIAGEN blood and tissue DNA isolation kit. The genomic DNA was prepared for sequencing using the KAPA HyperPlus Kit (Kapa Biosystems) and the resulting libraries were sequenced on an Illumina NextSeq platform. Fastq output files were imported into CLC Genomics Workbench software (QIAGEN) where all analysis was performed. Reads were trimmed and quality filtered before mapping of the reads, originating from the plasmid cured genomes, to the assembled control genomes. SNP and small INDEL variants were detected using quality based variant detection and larger INDELS and structural variants were assessed using the “Structural Variants and InDels” pipeline as well as by manual inspection of read mappings.
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3

Whole Genome Sequencing of ASFV

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Genohub Inc. (Austin, Texas USA) facilitated the whole genome sequencing service of the genomic DNA purified directly from ASFV-infected pig spleen using the Illumina HiSeq X platform (Illumina, USA). DNA Libraries were prepared using KAPA HyperPlus Kit (KAPA Biosystems, Wilmington, MA) according to the manufacturer’s protocol with 10 ng DNA as input. The final library quality and quantity were analyzed in a Bioanalyzer 2100 (Agilent Technologies, USA) and Qubit™ Fluorometer (Thermo Scientific, USA), respectively. Whole genome sequencing (WGS) of 150 bp paired-end reads were sequenced on Illumina HiSeq X (Illumina Inc., San Diego, CA). A total of 42 Gbp paired-end reads of 150 bp long were obtained.
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4

Amplification and sequencing of bacterial 16S rRNA and fungal ITS1 regions

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Total extracted DNA was used for high throughput sequencing (MiSeq platform, Illumina, San Diego, CA, USA) of the bacterial 16S rRNA gene or fungal ITS1 region. We amplified the V3–V4 region of 16S rRNA gene using degenerate barcoded primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′), and the ITS1 region using degenerate fungal primers ITS1-5.8Sfw (5′-CTGTAAAAGTCGTAACAAGGTTTC-3′) and ITS1-5.8Srv (5′-AAGTTCAAAGAYTCGATGATTCAC-3′). PCR amplifications (KAPA 2G Robust Hot Start DNA Polymerase, Kapa Biosystems, Hoffmann-La Roche, Switzerland) were carried out with 25 or 27 cycles, respectively. PCR products were purified and normalized with the SequalPrep™ Normalization Plate Kit (Thermo Fisher Scientific, Waltham, MA, USA). Triplicates of the amplicons were pooled, gel eluted (NucleoSpin gel clean-up, Macherey-Nagel, Düren, Germany), and ligated with sequencing adapters (TruSeq DNA PCR-free LT Sample Preparation Kit, Illumina; KAPA HyperPlus kit, Kapa Biosystems, Hoffmann-La Roche, Switzerland). Amplicon libraries were pooled in equimolar concentrations, validated by the KAPA Library Quantification Kit (Illumina, San Diego, CA, USA), and sequenced on the MiSeq platform using the 2 × 300 bp kit at the CEITEC Genomics Core Facility (CEITEC, Masaryk University, Brno, Czech Republic).
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5

Sequencing of KSHV Genome from SPEL Cells

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KSHV particles were collected from 75 mL of culture supernatant of 10 μM of suberic bishydroxamate (SBHA)-stimulated SPEL cells. The culture supernatant was subjected to ultracentrifugation at 100,000 × g for 2 h. The pellet was treated with DNase and RNase for 1 h. After heat inactivation at 70 °C for 10 min, viral DNA was extracted from the pellet. A DNA library was prepared with KAPA HyperPlus kit (Kapa Biosystems, Wilmington, MA, USA) with five cycles of PCR enrichment, followed by gel extraction for purification of the DNA library. Next-generation sequencing was performed using a MiSeq Reagent Kit v3 (600-cycle; Illumina, San Diego, CA, USA). Sequence reads were trimmed and assembled using the VirusTAP pipeline software (https://gph.niid.go.jp/cgi-bin/virustap/index.cgi) [28 (link)]. Sequence gap regions were amplified by conventional PCR, and sequenced using the Sanger method. The complete genome sequence of SPEL KSHV was deposited in the GenBank database under accession no. AP017458.
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6

Targeted NGS of Ube3a-ATS Locus

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We performed PCR from 5 ng genomic DNA isolated (Trizol) from the cortex of 10 month old mice dual injected with either negative gRNA (n=3), or Sajw33 (n=3). Multiple primer sets were designed to amplify all Sajw33 target sites across the Ube3a-ATS locus, and AAV integration events (Snord115 1 F, Snord115 4 F, Snord115 5 F, Snord115 1 R, Snord115 2 R, Snord115 3 R, ITR C R, ITR 2 R, AAV hSyn R). Off target primers are listed in Supplementary Table 6. PCR reactions were performed on the QuantStudio5 qPCR machine and halted once all samples reached exponential growth phase (cycle #26) in order to avoid over-amplification bias. Each PCR reaction was performed in triplicate to reduce random PCR sampling bias (360 PCRs total). PCRs from individual mice were pooled, PCR purified (Qiagen), end repaired and A-tailed (KAPA HyperPlus kit), and adaptors were ligated using KAPA dual-indexed adaptors ligation kit (KAPA Biosystems, Wilmington, MA). Libraries were purified using AMPure XP beads (Beckman Coulter, Brea, CA), and sequenced using the Illumina Miniseq system with Miniseq Mid Output kit (Illumina) (300 cycles), 150 bp paired end reads. Reads were filtered for adaptor contamination using cutadapt34 and filtered such that at least 90% of bases of each read had a quality score > 20.
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7

Bacterial Genomic DNA Extraction and Sequencing

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Isolates were grown overnight on blood agar plates at 37 °C in an atmosphere of 5 % CO2. Genomic DNA was extracted using an automated MagNAPure isolation station and MagNAPure 96 DNA and Viral NA small volume kit (Roche), according to the manufacturer's instructions. DNA concentration was measured using a Qubit fluorometer (Invitrogen, Thermo Fisher Scientific). Sequencing libraries were generated using a KAPA HyperPlus kit (KAPA Biosystems, Roche) and NEXTflex DNA Barcodes (Bioo Scientific), as previously described [21 (link)]. Clean-up and size selection were performed using magnetic AMPureXP beads (Beckman Coulter). DNA was then quantified using a Qubit fluorometer and the size of fragments was measured using an Agilent 2100 Bioanalyzer system (Agilent Technologies). Library samples were pooled and sequenced on the Illumina MiSeq platform with MiSeq reagent kits v2 500-cycles (Illumina).
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8

Metagenomic Library Construction and Illumina Sequencing

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The metagenomic library was constructed using KAPA HyperPlus Kit (KAPA Cat. NO. 0000097583, Boston, USA) with dual-indexed Adapters (KAPA Cat. NO. 0000093370, Boston, USA), the DNA was fragmented to 250 bp approximately by the enzyme at 37 °C for 20 min, after end repair and A-tailing, adapter ligation, post-ligation cleanup, library amplification, and post-amplification cleanup, the library was constructed.
Agilent 2100 Bioanalyzer (Agilent Technologies, Beijing, China) was used for library quality control, and qualified DNA library was sent to the Novogene company to sequence in HiSeq 4500 (Illumina, Beijing, China), and all raw data were trimmed by Trimmomatic (version 0.39) [22 (link)] to remove adapter sequence, low-quality reads, and duplicate reads.
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9

DNA Library Preparation and Sequencing

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One microgram of double-stranded DNA (dsDNA) was used for library synthesis using the KAPA Hyperplus kit (KAPA Biosystems, Wilmington, MA) with single-index adapters. In brief, dsDNA was enzymatically fragmented to an average of 200 base pairs. Fragments were purified using AmpureXP beads (Beckman Coulter, Brea, CA). Libraries were amplified for 69 cycles, quality checked on the LabChip GX Touch 24 Bioanalyzer (PerkinElmer, Waltham, MA), and quantified using the picogreen assay (Thermo Fisher Scientific) on the Victor X2 Fluorescent Microplate Reader (PerkinElmer). Completed libraries were pooled by equal mass for sequence capture. VirCapSeq-VERT was performed according to the Nimblegen SeqCap protocol (Roche, Basel, Switzerland) as described previously [11 (link)]. Postcapture libraries were purified and amplified before sequencing. To ensure sufficient depth of coverage (approximately 10 million unique sequence reads/sample), uniquely barcoded samples were pooled at a maximum of 20 libraries per pool (20-plex) and sequenced on a lane of HiSeq 4000 (Illumina, San Diego, CA).
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10

Purification and Sequencing of Cell-Free DNA

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Total circulating DNA was extracted from a volume of 250 μL of plasma from each sample using the QIAamp Circulating Nucleic Acid Kit (QIAGEN) according to the manufacturer’s instructions. And purified with AMPure XP SPRI paramagnetic beads (Beckman Coulter). Sequencing libraries were prepared from purified cfDNA using the KAPA HyperPlus Kit (Kapa Biosystems) with standard Illumina indexed adapters (IDT) as detailed in Sanders J. et al. (2019)47 (link). Sample libraries were characterized using the Agilent 4200 TapeStation System (High Sensitivity DNA Kit) and quantified by qPCR using the NEBNext® Library Quant Kit for Illumina® (New England Biolabs). Paired-end 2×150 bp sequencing was performed on a NovaSeq 6000 instrument (Illumina), and samples were pooled across all four lanes during sequencing.
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