Genomic DNA was sequenced with an Illumina MiSeq instrument at the New York University Genome Technology Center using paired-end chemistry (2 × 300 bp). The sequencing library was prepared using the PCR-free version of a KAPA DNA Library Preparation Kit (Kapa Biosystems, Wilmington, MA, United States). Overlapping paired reads were merged using FLASH version 1.2.11 (Magoc and Salzberg, 2011 (link)). The merged reads were trimmed of adapter sequences and low quality bases with Trim Galore! version 0.4.42 , which is a wrapper script for Cutadapt (Martin, 2011 (link)) and FastQC (Andrews, 2010 ). The optimal k-mer size was estimated with KmerGenie version 1.7 (Chikhi and Medvedev, 2014 (link)) and the draft genome was assembled de novo with Velvet version 1.2.10 (Zerbino and Birney, 2008 (link)). The assembled genome was initially annotated and inspected with the web-based RAST annotation service and SEED Viewer (Aziz et al., 2008 (link); Overbeek et al., 2014 (link)). The final annotation was completed with the National Center for Biotechnology Information’s (NCBI) Prokaryotic Genome Annotation Pipeline (PGAP) (Klimke et al., 2009 (link)).
Kapa dna library preparation kit
Manufactured by Roche
Sourced in United States
The KAPA DNA Library Preparation Kit is a set of reagents and protocols designed for the preparation of DNA libraries for next-generation sequencing applications. The kit provides the necessary components to perform key steps in the library preparation process, including DNA fragmentation, end-repair, A-tailing, and adapter ligation.
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Lab products found in correlation
Hiseq 3000 sequencing system, by Illumina (7 mentions)
Truseq dna library preparation kit, by Illumina (6 mentions)
Hiseq 2000, by Illumina (5 mentions)
Nimblegen, by Roche (5 mentions)
Qiaamp circulating nucleic acid kit, by Qiagen (5 mentions)
Hiseq 3000, by Illumina (4 mentions)
Biotinylated oligonucleotide probes, by Integrated DNA Technologies (4 mentions)
Dneasy blood kit, by Qiagen (4 mentions)
Dneasy blood tissue kit, by Qiagen (3 mentions)
Nextseq cn500, by Illumina (3 mentions)
Qiaamp dna mini kit, by Qiagen (3 mentions)
S2 instrument, by Covaris (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Novaseq 6000 platform, by Illumina (2 mentions)
Streptavidin dynabeads, by Thermo Fisher Scientific (2 mentions)
S2 ultrasonicator, by Covaris (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Hiseq platform, by Illumina (2 mentions)
Applied biosystems 7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Hiseq 2000 sequencing system, by Illumina (1 mentions)
40 protocols using kapa dna library preparation kit
1
Illumina-based De Novo Genome Assembly
2
Targeted Cancer Genomic Profiling
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Sequencing libraries of gDNA were constructed with the KAPA DNA Library Preparation Kit (Kapa Biosystems, Wilmington, MA, USA), followed the manufacturer’s protocol. Libraries were hybridized to custom-designed biotinylated oligonucleotide probes (Integrated DNA Technologies, Iowa, IA, USA). The gDNA from tumors and tumor-adjacent tissues and leukocyte were subjected to next-generation sequencing of 1,021 frequently mutated cancer genes and exons 2–3 of HLA genes (see Table S1 in the online supplement).40 (link) Next generation sequencing (NGS) was performed using the HiSeq 2000 Sequencing System (Illumina, San Diego, CA) with 2 × 101-bp paired-end reads. DNA from leukocyte was used as germline control.
The quality control process was applied in FASTQ data from tumors and TALs and control sample by removing the terminal adaptor sequences and low-quality reads from raw data. BWA41 (link) (version 0.7.12-r1039) was employed to align the clean reads to the reference human genome (hg19). Picard (version 1.98) was used to mark PCR duplicates. Realignment and recalibration were performed for these redundant reads using GATK (version 3.4–46-gbc02625).
The quality control process was applied in FASTQ data from tumors and TALs and control sample by removing the terminal adaptor sequences and low-quality reads from raw data. BWA41 (link) (version 0.7.12-r1039) was employed to align the clean reads to the reference human genome (hg19). Picard (version 1.98) was used to mark PCR duplicates. Realignment and recalibration were performed for these redundant reads using GATK (version 3.4–46-gbc02625).
3
Comprehensive Genomic Profiling of Samples
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Sequencing was carried out using Illumina 2 × 75-bp paired-end reads on an Illumina HiSeq 3000 instrument according to the manufacturer's recommendations using the KAPA DNA Library Preparation Kit (Kapa Biosystems, Wilmington, MA, USA). Bar-coded libraries were hybridized to a customized panel of 1,021 genes containing whole exons and selected introns of 288 genes and selected regions of 733 genes (Table S1 ). The libraries were sequenced to a uniform median depth (>500×) and assessed for somatic variants including single-nucleotide variants (SNVs), small insertions and deletions (InDels), copy number alterations (CNAs), and gene fusions/rearrangements.
4
Targeted Sequencing of Multiple Myeloma
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Each genomic DNA preparation extracted from SW and BM aspiration samples was cut into 200–250-bp fragments using a Covaris S2 instrument (Woburn, MA, USA) before library construction. After end-repair and polyA tailing, adapters with unique identifiers were ligated to both ends of double-stranded cfDNA fragments. Indexed Illumina Next Generation Sequencing (NGS) libraries were prepared from cfDNA, BM tumor DNA and SW germline DNA using the KAPA DNA Library Preparation Kit (Kapa Biosystems, Wilmington, MA, USA) following the manufacturer’s protocol. Target capture was performed using custom SeqCap EZ libraries (Roche NimbleGen, Madison, WI, USA). In order to explore the comprehensive genetic characteristics of multiple myeloma, the capture probe was designed based on 1.7 Mb genomic regions of 413 genes, involved in the oncogenesis and progression of hematological malignancies, such as multiple myeloma, lymphoma, etc. (Table S1 ). Capture hybridization was performed according to the manufacturer’s recommendations. The captured DNA fragments were then amplified and pooled to generate several multiplexed libraries. Sequencing was performed on the Illumina HiSeq 3000 instrument with 75 × 75 paired-end reads according to the manufacturer’s recommendations using an Illumina TruSeq PE Cluster Generation Kit v3 and the TruSeq SBS Kit v3 (Illumina, San Diego, CA, USA).
5
Targeted Sequencing of Solid Tumor Mutations
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Both cfDNA and gDNA libraries were constructed with the KAPA DNA Library Preparation Kit (Kapa Biosystems, Wilmington, MA, USA) using the manufacturer's protocol. Capture probes were designed to cover coding sequences and hot exons of 1021 genes that are frequently mutated in solid tumors. A detailed description of the capture experiments has been reported previously [28 (link)]. Libraries were hybridized to custom-designed biotinylated oligonucleotide probes (Integrated DNA Technologies, Iowa, IA, USA). DNA sequencing was performed using the HiSeq 3000 Sequencing System (Illumina, San Diego, CA) with 2 × 101-bp paired-end reads. In Table S2 , all genes included in our panel are listed. Clonal hematopoietic mutations were filtered as previously described [29 ], including those in DNMT3A, IDH1, and IDH2 and specific alterations within ATM, GNAS, and JAK2.
6
Chromatin Immunoprecipitation and Sequencing
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Chromatin Immunoprecipitation (ChIP) was performed as described previously [48 (link)] using αHDAC2 antibody from Bethyl laboratories (Cat#A300-705/Lot#1). Sequencing libraries were constructed from 0.2ng of input and immunoprecipitated DNA using the KAPA DNA Library Preparation Kit (KapaBiosystems). Sequencing on an Illumina HiSeq 2000 sequencer yielded between 13 and 30 million reads. Analysis of sequence data was as previously described [49 (link)], except that the genome was tiled into 50 bp windows.
7
Comprehensive Genomic Profiling of OCCC
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A tissue kit was used to extract genomic DNA (gDNA) from OCCC samples and matched normal tissues (Qiagen, Hilden, Germany). Following the manufacturer’s instructions, sequencing libraries were created using the KAPA DNA Library Preparation Kit (Kapa Biosystems, MA, USA). For both biopsy samples, barcoded libraries were hybridized to a panel of 1,021 genes with full exons, chosen introns from 288 genes, and selected regions from 733 genes. The comprehensive gene list was described in our earlier research (Wang et al., 2020 (link)). The DNBSEQ-T7RS (BGI, Shenzhen, China) with 100 bp paired-end reads was used to sequence the DNA.
8
Targeted DNA Sequencing of Circulating and Tissue Samples
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In this study, a 727-gene panel covering a 2.8 megabase (Mb) region was designed to capture the target DNA fragments. The lower limit of the target panel detection was ≥ 0.50% for somatic single nucleotide variants (SNVs) and small insertions/deletions (InDels) and ≥ 3 copies for copy number variants (CNVs). The list of genes detected in this study is presented in Supplementary Table 1 . All of the collected cfDNA from C1 and C2 and tDNA from tissue samples were subjected to DNA library preparation using a Kapa DNA library preparation kit (Kapa Biosystems, Wilmington, USA) while the gDNA library was prepared using an Illumina TruSeq DNA library preparation kit (Illumina, San Diego, USA). The DNA libraries were hybridized to our custom-designed 727-gene probes (Nanodigmbio, Nanjing, China). Then, DNA sequencing was conducted using a DNBSEQ-2000 sequencer (BGI, Shenzhen, China). The raw sequencing data was filtered according to the default parameters to retain high quality reads for subsequent analysis. The clean reads were aligned to human genome assembly (hg19) using the Burrows-Wheeler Aligner (BWA-0.7.17-r1188). Removal of duplicate reads, local realignments, and base-quality recalibrations were performed using the Genome Analysis Toolkit (GATK 4.0.12.0). The resulting binary alignment map (BAM) files were used for subsequent variant calling analysis.
9
Single-cell whole-genome DNA amplification and sequencing
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Re-suspended cells were lysing and whole-genome DNA amplified using the REPLI-g Single Cell Kit (Qiagen) using the manufacturer’s recommended protocol. Amplified DNA was purified by AMPure XP beads (Beckman Coulter, Brea, CA) using the manufacturer’s recommended protocol resulting in 25 µL of purified WGA product.
Purified WGA products were sheared to generate DNA fragments averaging 350 bps by sonication (Covaris, Woburn, MA). Sonicated DNA was cleaned, end-repaired, ligated, and amplified using the KAPA DNA Library Preparation Kit (KAPA Biosystems, Wilmington, MA) according to the manufacturer’s protocol. Sequencing was performed on an Illumina NextSeq 500 (Illumina, San Diego, CA) using 75 bp paired end reads (2 × 75 bp).
Purified WGA products were sheared to generate DNA fragments averaging 350 bps by sonication (Covaris, Woburn, MA). Sonicated DNA was cleaned, end-repaired, ligated, and amplified using the KAPA DNA Library Preparation Kit (KAPA Biosystems, Wilmington, MA) according to the manufacturer’s protocol. Sequencing was performed on an Illumina NextSeq 500 (Illumina, San Diego, CA) using 75 bp paired end reads (2 × 75 bp).
10
DNA Library Preparation and Sequencing
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The Illumina compatible libraries were prepared using KAPA DNA Library preparation kit (Catalog No. KK8232) as per the manufacturer’s protocol. In brief, DNA was fragmented to a median size of 200bp by sonication. Fragmented DNA ends were polished and 5′-phosphorylated. After addition of 3′-A to the ends, indexed Y-adapters were ligated and the samples were PCR amplified. The resulting DNA libraries were enriched for targeted regions using NimbleGen SeqCap EZ Choice Library 4 RXN (Catalog No. 06740251001) and NimbleGen SeqCap EZ Reagent Kit Plus v2 (Catalog No. 06953247001) as per the manufacturer’s protocol. The enriched libraries were quantified and validated by qPCR, and sequenced on Illumina’s HiSeq 2000 in a paired-end read format for 76 cycles. The resulting BCL files containing the sequence data were converted into “.fastq.gz” files and individual sample libraries were demultiplexed using CASAVA version_1.8.2 with no mismatches.
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