Forty μl of total nucleic acid was extracted from 240 μl of stool suspension using the NucliSENS easyMAG automated platform (BioMérieux, Boxtel, The Netherlands). Individual VirCapSeq-VERT libraries were prepared using the Hyper Prep kit (KAPA Biosystems, Boston, MA, USA) and unique barcodes. Superscript III and random hexamer primers were used to generate first strand cDNA (Life Technologies, Carlsbad, CA, USA). Second-stranded cDNA synthesis for HTS was carried out by random primer extension with Klenow enzyme (New England Biolabs, Ipswich, MA, USA). Double stranded DNA preparations were sheared (E210 sonicator; Covaris, Woburn, MA, USA) for an average fragment size of 200 base pairs and added to Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) for purification, and libraries were prepared with the Hyper Prep kit (KAPA Biosystems, Wilmington, MA, USA). Libraries were pooled and hybridized with the VirCapSeq-VERT probe set prior to a final PCR and sequencing (Illumina HiSeq 4000).
Hyper prep kit
Manufactured by Roche
Sourced in United States
The Hyper Prep Kit is a laboratory equipment product designed for the preparation of sequencing libraries. It is a tool used in the process of preparing DNA or RNA samples for sequencing analysis.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2500, by Illumina (12 mentions)
Hiseq x, by Illumina (4 mentions)
Nextseq 500, by Illumina (4 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Hifi hotstart readymix, by Roche (3 mentions)
Hiseq 4000, by Illumina (3 mentions)
Nextseq high output flow cell, by Illumina (3 mentions)
Anti scp3, by Santa Cruz Biotechnology (2 mentions)
Anti h3k4me3, by Merck Group (2 mentions)
Anti dmc1 santa, by Santa Cruz Biotechnology (2 mentions)
Anti dmc1, by Merck Group (2 mentions)
Epitect bisulfite kit, by Qiagen (2 mentions)
Hiseq 4000 platform, by Illumina (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Bioanalyzer high sensitivity dna kit, by Agilent Technologies (2 mentions)
Nextseq high output flow cell 300 cycles pe150, by Illumina (2 mentions)
Allprep dna rna mini kit, by Qiagen (2 mentions)
2.0 fluorometer, by Illumina (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Nextseq 500 instrument, by Illumina (2 mentions)
78 protocols using hyper prep kit
1
Viral Enrichment from Stool Samples
2
Whole Genome Bisulfite Sequencing
3
Epigenomic profiling of induced T regulatory cells
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Viable iTreg cells were sorted by Foxp3gfp reporter expression after induction. Cut&Run was performed as described (Skene and Henikoff, 2017 (link)). Briefly, 1 × 106 iTreg cells were first attached to Concanavalin A (ConA)-coated magnetic beads, permeabilized with digitonin-wash buffer (20 mM HEPES, pH7.5, 150 mM NaCl, 0.5 mM spermidine, 0.01% digitonin, and protease inhibitors), and then incubated with antibody (1:100 H3K27ac) for 2 h at 4°C on a rotator. The beads-cell mixture was washed thrice with digitonin-wash buffer, resuspended in 200 μL of protein A-MNase, and incubated for 1 h at 4°C on a rotator. After 3 rounds of washing, beads were resuspended in 150 μL of digitonin-wash buffer and chilled in an ice-water bath for 5 min; 3 μL of 100 mM CaCl2 was added into the tubes with gentle vortexing, and the beads were incubated in an ice-water bath for 30 min. Next, 150 μL of 2 × STOP buffer (170 mM NaCl, 20 mM EDTA, 20 mM EGTA, 0.05% digitonin, 20 mg/mL GlycoBlue, 25 mg/mL RNase A) was added and incubated at 37°C for 30 min. After clarification on a magnet stand, supernatant was transferred to a fresh tube for phenol:chloroform extraction and ethanol precipitation. A library was prepared by using KAPA Hyper Prep Kits, and 5 ng DNA was loaded for high-throughput sequencing.
4
Comprehensive Genomic Profiling for Research
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DNA samples were used for two different assays including whole exome sequencing (WES) and low pass long insert whole genome sequencing (WGS). WES was prioritized if material was limiting. For WES, 50ng-250ng of genomic DNA was fragmented to an average size of 180bp in length using a Covaris focused-ultrasonicator (Covaris). An Illumina sequencing technology compatible whole genome library was created using Kapa Biosystems Hyper Prep Kits. These libraries were then subjected to whole exome target enrichment using Agilent SureSelect V5+UTR hybrid capture kits.
For RNA-sequencing, either 150ng or 500ng of total RNA was used to enrich for poly-adenylated RNA molecules, which were subsequently fragmented to a target size of 180bp by heat fragmentation. Fragmented molecules were then converted to cDNA using random primers with Superscript II (Invitrogen). After second strand synthesis, the resulting molecules were used for library prep using the Illumina TruSeqRNA library kit.
For RNA-sequencing, either 150ng or 500ng of total RNA was used to enrich for poly-adenylated RNA molecules, which were subsequently fragmented to a target size of 180bp by heat fragmentation. Fragmented molecules were then converted to cDNA using random primers with Superscript II (Invitrogen). After second strand synthesis, the resulting molecules were used for library prep using the Illumina TruSeqRNA library kit.
5
Epigenomic profiling of induced T regulatory cells
6
Comprehensive Genomic Profiling of Solid Tumors
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After expert pathologist assessment, solid tumor genomic DNA was isolated from formalin-fixed, paraffin-embedded tumor samples. DNA libraries were prepared using the KAPA Hyper Prep Kit, hybridized to the xT probe set, and amplified with the KAPA HiFi HotStart ReadyMix. The amplified target-captured DNA tumor libraries were sequenced to an average target depth of 500 × on an Illumina HiSeq 4000 using the Tempus xT (Tempus labs Chicago, IL, USA) gene panel. The panel analyzes single nucleotide variants (SNVs), indels, and copy number variants in 596 genes and genomic rearrangements in 21 genes with an average coverage of 500x28 (link). Variants were called using Freebayes (version 1.0.2). Clinically relevant mutations were identified using the Cancer Genome Interpretor29 (link) and ClinVar30 (link). Data was graphed using Microsoft Excel 2016.
7
Oocyte H3K4me3 ChIP-Seq Protocol
8
Metagenomic sequencing of HHV-6 genomes
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DNA was extracted from culture isolates using the Zymo viral DNA kit. DNA-sequencing libraries were prepared from 50 ng of genomic DNA using quarter volumes of the Kapa HyperPrep kit with 7 min of fragmentation time and 12 cycles of dual-indexed TruSeq adapter PCR (20 (link)). The libraries were sequenced on 2 × 300-bp, 1 × 190-bp, and/or 1 × 192-bp runs on an Illumina MiSeq. The sequences were quality and adapter trimmed using BBDuk (http://jgi.doe.gov/data-and-tools/bbtools/ ) and de novo assembled using SPAdes (58 (link)), and contigs were aligned with reference HHV-6A (NC_001664 ) and HHV-6B (NC_000898 ) genomes and visualized using Geneious v9.1. Read mapping for copy number analysis was performed against the HHV-6A (NC_001664 ) and HHV-6B (NC_000898 ) reference genomes, using the Geneious read mapper with 10% allowed gaps per read, word length of 18, and 20% maximum mismatches per read and with structural variant, insertion, and gap finding allowed.
9
WGBS Library Preparation and Analysis
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For the preparation of WGBS libraries, genomic DNA was first extracted from leaves and inflorescence tissue (for Ler samples) using the DNeasy Plant Mini Kit (Qiagen). 100 ng of DNA was then used for subsequent shearing using a Covaris S2 Focused Ultrasonicator. Libraries were then prepared using either the Ovation Ultralow Methyl-Seq kit (NuGen) in conjuction with the EpiTect Bisulfite Kit (Qiagen), or the Hyper Prep Kit (KAPA Biosystems) in conjuction with either the EZ DNA Methylation-Lightning Kit (Zymo) or the EpiTect Bisulfite Kit (Qiagen). Single-end 50 bp reads were then uniquely aligned to the TAIR10 genome using BS-Seeker258 (link). Methylation levels were then calculated for the CG, CHG, and CHH contexts. A filter was implemented to remove reads with three or more consecutively methylated cytosines in the CHH context, as previously described59 (link). Metaplots of BS-seq data were generated with custom Python and R scripts. For methylation calculations over individual chromosomes, each chromosome was split into 100 kb bins. Methylation values were then calculated from these bins.
10
Distinguishing Viral Sequence Origins in Agave
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In order to distinguish whether viral sequences were derived from an endogenous or exogenous origin, we sequenced three (one for each Agave taxa) DNA libraries from the leaves of the same individuals from which we produced the transcriptomes. Genomic DNA was prepared using the KAPA Hyper Prep Kit (07962312001) following the manufacturer’s instructions. For the library preparation, 1 ug of fragmented DNA with an average length of 280 bp was used as input material, and then four PCR cycles were used for amplification. Libraries were sequenced with Illumina HiSeq 4000 platform Finally, to investigate the origin of possible viral sequences, raw genomic reads were aligned to the candidate viral genomes using Bowtie2 allowing zero mismatches. Removal of false-positive results was adapted from Aguiar et al. (2015) [42 (link)]. Briefly, we removed sequences presenting similarity with retroviral elements, containing truncated ORFs and viral transcripts assigned to non-retroviral families that presented alignment from DNA sequencing libraries (at least 10 reads covering 70% sequence) were considered derived from endogenous viral elements (EVEs).
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