For whole genome sequencing of AP3_16T strain, DNA was isolated following a previously described protocol (Furmanczyk et al., 2017a (link)). The isolated genomic DNA was used to prepare two types of libraries: (1) paired-end library with average insert size 500 bp (using KAPA HTP Library Preparation Kit for Illumina platforms according to manufacturer’s protocol) (Kapa Biosystems, United States), (2) Nextera® Mate Pair library with average insert size 8 kbp (using Illumina protocol) (Illumina, United States). The libraries were verified using a 2100 Bioanalyzer (Agilent, United States) High-Sensitivity DNA Assay and KAPA Library Quantification Kit for the Illumina (Kapa Biosystems, United States). Sequencing was performed using an Illumina MiSeq (MiSeq Reagent Kit v3, 600 cycles) (Illumina, United States) with read length of 300 bp.
Kapa htp library preparation kit
Manufactured by Illumina
Sourced in United States
The KAPA HTP Library Preparation Kit is a high-throughput library preparation solution for next-generation sequencing. The kit provides reagents and protocols for the construction of DNA libraries from a variety of input materials, including genomic DNA, PCR amplicons, and cDNA.
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Lab products found in correlation
Novaseq 6000, by Illumina (2 mentions)
Hiseq x ten system, by Illumina (2 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nextera mate pair library, by Illumina (1 mentions)
High sensitivity dna assay, by Illumina (1 mentions)
Tianamp genomic dna kit, by Tiangen Biotech (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Tguide s32 magnetic blood genomic dna kit, by Tiangen Biotech (1 mentions)
Accugreen high sensitivity dsdna quantitation kit, by Biotium (1 mentions)
Magmax cell free dna isolation, by Thermo Fisher Scientific (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
H3k27ac, by Diagenode (1 mentions)
H3k4me1, by Cell Signaling Technology (1 mentions)
H3k36me3, by Diagenode (1 mentions)
H3k27me3, by Cell Signaling Technology (1 mentions)
H3k9me3, by Abcam (1 mentions)
H3k4me3, by Cell Signaling Technology (1 mentions)
Le220 focused ultrasonicator, by Covaris (1 mentions)
Hiseq x ten platform, by Illumina (1 mentions)
8 protocols using kapa htp library preparation kit
1
Whole Genome Sequencing of AP3_16^T Strain
2
Comprehensive Genomic Profiling of FFPE Tumors
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DNA was isolated from formalin-fixed paraffin-embedded (FFPE) tumor specimens with the TIANamp genomic DNA kit (TIANGEN, China) according to the manufacturers’ instructions. Genome DNA is extracted by TGuide S32 magnetic blood genomic DNA kit (TIANGEN, China) from peripheral blood lymphocyte (PBL), and circulating cell-free DNA (cfDNA) is extracted by MagMAX cell-free DNA isolation (ThermoFisher, USA) from the plasma sample. Fragmented DNA libraries were constructed with a KAPA HTP library preparation kit (Illumina Platform) (KAPA Biosystems, Massachusetts, USA) according to the manufacturer’s instructions. All libraries were quantified using AccuGreen high sensitivity dsDNA quantitation kit (Biotium, USA), with library size assessed on agilent bioanalyzer 2100 (Agilent, USA). DNA libraries from baseline tissue samples were captured with Panel 1, which was a designed panel spanning 769 cancer-related genes (Genecast, Wuxi, China), while DNA libraries from plasma samples were captured with panel 2, which covered exon regions of 95 genes (Genecast, Wuxi, China) related to drug resistance. The captured library was sequenced on Illumina Novaseq 6000 with paired end 150 bp mode.
3
Targeted Sequencing of Cancer Genes
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Genomic DNA was sheared into 150–200 base pairs (bp) fragments. Fragmented DNA libraries were constructed by KAPA HTP Library Preparation Kit (Illumina platforms; KAPA Biosystems) following producer’s instruction.
DNA was hybridized to two designed Genescope panels: (1) 543 genes (Genecast) that included tumor-related major genes, covering 1.7 Mb of the genome, and (2) 773 genes (Genecast) that included tumor-related major genes, covering 1.9 Mb of the genome. The final sequencing libraries were quantified using Qubit dsDNA HS Assay Kit (Thermo Fisher). The captured samples were subjected to Illumina Novaseq6000 for paired end sequencing.
DNA was hybridized to two designed Genescope panels: (1) 543 genes (Genecast) that included tumor-related major genes, covering 1.7 Mb of the genome, and (2) 773 genes (Genecast) that included tumor-related major genes, covering 1.9 Mb of the genome. The final sequencing libraries were quantified using Qubit dsDNA HS Assay Kit (Thermo Fisher). The captured samples were subjected to Illumina Novaseq6000 for paired end sequencing.
4
Chromatin Immunoprecipitation Protocol
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The following antibodies were used for chromatin immunoprecipitation: (i) H3K4me1: Cell Signaling Technologies, cat #5326BF, lot #2 (quantity: 3 µg/concentration: 0.015 µg/µl); (ii) H3K4me3: Cell Signaling Technologies, cat #9751BF, lot#6 (quantity: 5 µg/concentration: 0.025 µg/µl); (iii) H3K9me3: Abcam, cat #Ab8898, lot #GR93671-1 (quantity: 3 µg/concentration: 0.015 µg/µl); (iv) H3K27me3: Cell Signaling Technologies, cat #9733S, lot #6 (quantity: 10 µg/concentration: 0.05 µg/µl); (v) H3K27ac: Diagenode, cat #pAB-196-050, lot #A1723-0041D (quantity: 6 µg/concentration: 0.03 µg/µl); (vi) H3K36me3: Active motif, cat #MABI0333, lot #12003 (quantity: 2 µg/concentration: 0.01 µg/µl). Libraries were prepared using the automated protocol for the Kapa HTP Library Preparation Kit (Illumina), and sequencing was performed using the Illumina HiSeq 2000, as per the manufacturer’s instructions, to achieve at least 30 and 60 million reads for narrow (H3K27ac and H3K4me3) and broad (H3K27me3, H3K36me3, H3K4me1, and H3K9me3) marks, respectively (Supplementary Fig. 1a ).
5
Targeted Sequencing of Genomic DNA
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Genomic DNA was sheared to ~ 150- to 200-bp average size by using a Covaris LE220 focused ultrasonicator. The fragmented DNA was then end-repaired, dA-tailed, adapter-ligated, and enriched by PCR amplification using Kapa HTP library preparation kit Illumina 96rxn. Designed baits were hybridized with adapter-ligated DNA libraries for 64 to 72 h. Then, the bait-target hybrids were captured by streptavidin beads and enriched via secondary PCR enrichment. The capture libraries were sequenced by performing paired-end 150 cycles on the Illumina HiSeq X Ten system at 50,000X. This dataset has a median of 87,094 (range 31,437–129,934) base pairs covered at ≥ 15,000X across 47 samples (see detailed sample list in Additional file 1 : Table S4).
6
Targeted Sequencing of SLC31A1 in Bladder Cancer
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The genomic DNA from a subset of 10 bladder cancer samples was subjected to fragmentation, resulting in fragments of 150–200 bp in size. This fragmentation process was carried out using the Covaris M220 Focused-ultrasonicator™ Instrument (Covaris, Massachusetts, USA). Subsequently, libraries for the fragmented DNA and were prepared using the KAPA HTP Library Preparation Kit (Illumina platforms) from KAPA Biosystems (Massachusetts, USA), following the manufacturer’s instructions. For the targeted sequencing of the SLC31A1 gene, a custom Genescope panel (Genecast, Beijing, China) was employed. The prepared DNA libraries were then subjected to a capture process using this panel. The captured samples were subsequently subjected to paired-end sequencing on an Illumina HiSeq X-Ten platform. Finally, the observed genetic mutations were interpreted according to the American College of Medical Genetics and Genomic (ACMG) guidelines16 (link)
and annotated by utilizing the ClinVar database.17
and annotated by utilizing the ClinVar database.17
7
EGFR Hotspot Mutation Detection via Ultrasensitive NGS
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Target enrichment was performed using amplicon-based method adopting custom designed amplicons for the EGFR Exon 19/Exon 21 region. The targeted region consists of recurrent hotspot mutations (including SNPs, insertions/deletions less than 25 bp) in EGFR. Input of ctDNA varied in the range of 3–50 ng for target enrichment. The amplicons were subjected to NGS library preparation using KAPA HTP library preparation kit from Illumina. The NGS libraries were sequenced at ultra-high depth of 100,000× depth using Illumina (San Diego, CA, USA) HiSeq 2500® instrument.
8
Targeted Sequencing of Tumor Genes
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Genomic DNA was sheared into 150–200 bp fragments using a Covaris M220 Focused-ultrasonicator (Covaris, Massachusetts, USA). Once the fragment size met the requirements, the KAPA HTP Library Preparation Kit (Illumina platform, KAPA Biosystems, Massachusetts, USA) was applied to construct the DNA Library according to the manufacturer’s instructions. The DNA library was captured with a designed 1406-gene panel (Genecast, Beijing, China) that included major tumor-related genes. The captured samples were then subjected to a HiSeq X-Ten system (Illumina, San Diego, California, USA) for paired-end sequencing.
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