Ultrasound was used to fragment cDNA in Snap-Cap microTUBEs at 4°C using a Covaris S220 (Woburn, MA, USA). The fragmentation conditions were as follows; run time 55 s, peak power 175.0 W, duty factor 5.0% and 200 cycles/burst. The Illumina library was constructed with KAPA Hyper Prep Kit Illumina platforms (Kapa Biosystems, Woburn, MA, USA). The quantity of the library was evaluated using the KAPA library quantification kit (Kapa Biosystems). Each 300 bp of the paired-end sequences of each fragment were determined with the Illumina MiSeq platform (San Diego, CA, USA).
Kapa hyper prep kit platform
Manufactured by Illumina
Sourced in United States
The KAPA Hyper Prep Kit is a library preparation kit designed for use with Illumina sequencing platforms. The kit provides reagents and protocols for the construction of high-quality sequencing libraries from a variety of input DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Miseq platform, by Illumina (3 mentions)
Miseq reagent kit v3, by Illumina (3 mentions)
Kapa library quantification kit, by Roche (2 mentions)
Smarter race 5 3 kit, by Takara Bio (2 mentions)
Novaseq 6000 pe150, by Illumina (2 mentions)
Smarter universal low input rna kit, by Takara Bio (1 mentions)
Hiseq machine, by Illumina (1 mentions)
Magmax cell free dna isolation kit, by Thermo Fisher Scientific (1 mentions)
Dnase 1 amplification grade, by Thermo Fisher Scientific (1 mentions)
S1 nuclease, by Thermo Fisher Scientific (1 mentions)
Nextseq 500 platform, by Illumina (1 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
8 protocols using kapa hyper prep kit platform
1
Illumina Library Construction from cDNA
2
Sequencing of dsRNA and ssRNA Libraries
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cDNA libraries were constructed from purified dsRNA and ssRNA as described previously (Urayama et al., 2018 (link)). In brief, dsRNA obtained from each sample was converted into a cDNA library using the FLDS method. The U2 primer was ligated to the 3′ end of fragmented dsRNA, and cDNA was synthesized using the SMARTer RACE 5′/3′ Kit (Takara Bio) with the U2-comp primer. Regarding total RNA-seq, ssRNA was converted into a cDNA library using the SMARTer Universal Low Input RNA Kit according to the manufacturer’s protocol (Takara Bio). After PCR amplification, cDNA was fragmented by an ultrasonicator (Covaris S220). Illumina sequencing libraries were then constructed using KAPA Hyper Prep Kit Illumina platforms (Kapa Biosystems) and evaluated using the KAPA Library Quantification Kit (Kapa Biosystems). The libraries were sequenced using the Illumina MiSeq v3 Reagent Kit (600 cycles) with 300-bp paired-end reads on the Illumina MiSeq platform.
3
Next-Gen Sequencing of Liquid Biopsy
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Blood instead of tissues was used for next-generation sequencing (Shanghai Tongshu Biotechnology Co, Ltd, Shanghai, China) because of few tissues available. We performed cell-free DNA extraction from the blood sample using the MagMAX Cell-Free DNA Isolation Kit (A29319; Applied Biosystems, Waltham, Mass). We created targeted capture pulldown and a targeted library from native DNA using a targeted gene sequencing panel, which covers 156 genes and KAPA Hyper Prep Kit Illumina platforms (#KR0961; Kapa Biosystems, Wilmington, Mass), and generated paired-end sequence data using Illumina HiSeq machines. The sequence data, aligned to the human reference genome (NCBI build 37) using BWA, and sorted and removed PCR duplication using GATK 4.0.11 (link) Somatic mutation calling was performed using VarDict 1.5.8.12 (link) Somatic mutations existing in at least 2 of the results of the 3 software were selected as high confident mutations. Copy-number variations (CNVs) and loss of heterozygosity were analyzed using CNVkit 0.9.6.dev0.13 (link)The results of next-generation sequencing showed alterations in ERBB2 with CNV amplification (20%) and mutations in exon19 (0.14%) and exon20 (0.12%). Mutations were also occurred in UGTIA1, GSTPI, and MTHFR. TMB (14.9 mutations/Mb) was much higher than the average (2.5 mutations/Mb).
4
Extraction and Sequencing of Viral dsRNA from Nori and Conchocelis Samples
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Two nori sheets (N1 and N2) and six conchocelis samples (C1–6) were disrupted using liquid nitrogen in a mortar, and total nucleic acids were manually extracted from the samples using SDS-phenol. dsRNA was then purified using cellulose resin chromatography and subjected to agarose gel electrophoresis. To obtain sequence-grade dsRNA, the remaining DNA and ssRNA were removed using amplification-grade DNase I (Invitrogen) and S1 nuclease (Invitrogen).
The resulting sequence-grade dsRNA was converted into cDNA using the FLDS method (Urayama et al., 2016 (link); Hirai et al., 2021 (link)). Briefly, dsRNA was fragmented using an ultrasonicator (Covaris S220; Covaris), adapter ligated (3′ ends), and converted to full-length cDNA using the SMARTer RACE 5′/3′ Kit (Takara Bio) with an adapter-complementary oligonucleotide primer. After PCR amplification, cDNA was fragmented using an ultrasonicator (Covaris S220), and Illumina sequencing libraries were constructed using the KAPA Hyper Prep Kit Illumina platforms (Kapa Biosystems). The resultant libraries were sequenced using the Illumina MiSeq v3 Reagent Kit (600 cycles) with 300-bp paired-end reads on the Illumina MiSeq platform.
The resulting sequence-grade dsRNA was converted into cDNA using the FLDS method (Urayama et al., 2016 (link); Hirai et al., 2021 (link)). Briefly, dsRNA was fragmented using an ultrasonicator (Covaris S220; Covaris), adapter ligated (3′ ends), and converted to full-length cDNA using the SMARTer RACE 5′/3′ Kit (Takara Bio) with an adapter-complementary oligonucleotide primer. After PCR amplification, cDNA was fragmented using an ultrasonicator (Covaris S220), and Illumina sequencing libraries were constructed using the KAPA Hyper Prep Kit Illumina platforms (Kapa Biosystems). The resultant libraries were sequenced using the Illumina MiSeq v3 Reagent Kit (600 cycles) with 300-bp paired-end reads on the Illumina MiSeq platform.
5
Illumina MiSeq RNA Sequencing Protocol
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Illumina sequencing libraries were then constructed using KAPA Hyper Prep Kit Illumina platforms (Kapa Biosystems) from the physically shared environmental cDNAs. The libraries were sequenced using the Illumina MiSeq v3 Reagent kit (600 cycles) with 300-bp paired-end reads on the Illumina MiSeq platform.
6
Genomic DNA Extraction and Sequencing from Snail Tissues
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Genomic DNA was extracted from a single iM line or iBS90 snail (the intestine excluded) using CTAB62 (link). DNA quality was assessed by agarose gel (1.0%) electrophoresis. Concentration and purity of the extracted DNA were evaluated using a Nanodrop 2000 spectrophotometer and a Qubit 3.0 Fluorometer (ThermoFisher Scientific). After selection of size (400–700 bp) using Blue Pippin, a 150 nt x 2 paired-end library was prepared (KAPA Hyper Prep Kit Illumina platforms, KAPA Biosystems, www.kapabiosystems.com ) and sequenced on the Illumina NextSeq500 platform at the University New Mexico (UNM) Biology Department’s Molecular Biology Facility (http://ceti.unm.edu/core-facilities/molecular-biology.html ).
7
FLDS: Novel dsRNA Sequencing Protocol
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Purified dsRNA was subjected to a novel method of library construction called FLDS [22 (link)]. This method consists of physical fragmentation of dsRNA following cellulose column chromatography, synthesis of complementary (c) DNA by reverse transcription (RT) using a modified rapid amplification of cDNA ends (RACE) method, and library construction and amplification of cDNA via PCR. Finally, dsRNA sequencing was performed using the KAPA Hyper Prep kit Illumina Platform (Kapa Biosystems, Woburn MA, USA). Illumina NovaSeq 6000/PE150 was used to determine 150 bp of the paired end sequence of each fragment. Raw sequencing data were processed through the FLDS pipeline (available in GitHub). CLC Genomic Workbench version 11.0 was used for de novo assembly of contigs >300 nt (other options were default). Full-length recovery of dsRNA segments was confirmed by manual analysis using CLC Genomic Workbench version 11.0, Genetyx version 14 and Tablet viewer version 1.19.09.03.
8
FLDS Method for Viral Genome Sequencing
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A novel method FLDS was used for the viral genome sequencing as described (Hirai et al., 2021 (link); Urayama et al., 2016 (link)).This method consists of physical fragmentation of dsRNA following cellulose column chromatography, synthesis of cDNA by reverse transcription (RT) using a modified RACE method, library construction and amplification of cDNA via PCR. Finally, dsRNA sequencing was done using the KAPA Hyper Prep Kit Illumina Platform (Kapa Biosystems, Woburn MA, USA). Illumina NovaSeq 6000/PE150 was used to determine 150 bp of the paired end sequence of each fragment. Raw sequencing data were processed through the FLDS pipeline (available in GitHub, https://github.com/takakiy/FLDS ). CLC Genomic Workbench version 11.0 was used for de novo assembly of contigs >300 nt (other options were default). Full-length recovery of dsRNA segments was confirmed by manual analysis using CLC Genomic Workbench version 11.0, Genetyx version 14, and Tablet viewer version 1.19.09.03.
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