Libraries were prepared with the use of an automated pipetting station (Biomek i5; Beckman Culter, Indianapolis, IN, USA). For library construction, 100 ng of genomic DNA was enzymatically fragmented with the use of a KAPA Frag Kit (Roche) for 15 min at 37 °C. Then, the procedure was continued with a KAPA HypPlus kit (Roche, Basel, Switzerland) and KAPA DI Adapter Kit (Roche) according to the manufacturer’s recommendation. The quality and quantity of libraries were confirmed by capillary gel electrophoresis (DNF-473 Standard Sensitivity NGS Fragment Analysis Kit, Fragment Analyzer, Agilent, Santa Clara, CA, USA) and fluorimeter (BR/HS assay kit, Qubit 3.0, Thermo Fisher Scientific).
Kapa frag kit
Manufactured by Roche
Sourced in United States
The KAPA Frag Kit is a laboratory tool designed for DNA fragmentation. It provides a standardized process for shearing DNA samples into smaller fragments, which is a crucial step in various genomic applications such as next-generation sequencing library preparation.
Automatically generated - may contain errors
Lab products found in correlation
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Tru q0, by Horizon Discovery (1 mentions)
Ultrasonicator, by Covaris (1 mentions)
Trusight oncology 500 kit, by Illumina (1 mentions)
Novaseq 6000 sequencer, by Illumina (1 mentions)
Ion s5, by Thermo Fisher Scientific (1 mentions)
Smart seq v4 ultra low input rna kit for sequencing, by Takara Bio (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Trusight oncology 500 assay, by Illumina (1 mentions)
Ffpe qc kit, by Illumina (1 mentions)
Nextseq 550 sequencer, by Illumina (1 mentions)
Nebnext multiplex oligo, by Illumina (1 mentions)
Accel ngs methyl seq dna library kit, by Integrated DNA Technologies (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
7 protocols using kapa frag kit
1
Automated Library Preparation for NGS
2
Synthetic cfDNA Library Preparation
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Reference material was purchased from Horizon Discovery, Tru-Q 4 (HD731) containing 5% allele frequency mutations, Tru-Q 7 (HD734) containing 1.3% allele frequency mutations, and a wild type sample Tru-Q 0 (HD752). In order to create a 0.13% allele frequency sample, the 1.3% material was diluted 1:10 in the WT material. To create a fragmented DNA analogue with a size distribution similar to that of cfDNA, the 5%, 1.3%, and 0.13% samples were independently fragmented using the KAPA Frag kit (Roche; 07962517001) following the manufacturers recommended conditions followed by a 3x AMPure bead purification. FFPE samples were either enzymatically fragmented as above or sonicated using a Covaris ultrasonicator. The size distribution was measured using an Agilent Bioanalyzer High Sensitivity kit and concentrations were determined using a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific; Q32854). The final panel targeted 169 conserved exons found as fusion partners across 22 genes. Details of all target genes are in Supplementary Table 5.
3
Comprehensive NGS Mutation Analysis of FFPE Samples
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For mutation analysis, TruSight Oncology 500 panel, a comprehensive NGS assay on FFPE samples that identifies fusion transcripts, somatic variants, copy number changes, tumor mutational burden, and microsatellite instability was used. NA libraries were created using the TruSight Oncology 500 Kit (Illumina) following the manufacturer’s instructions (KAPA Biosystems, Washington, MA), although we used KAPA FragKit (KAPA Biosystems, Washington, MA) for DNA enzymatic fragmentation. Following the manufacturer’s guidelines, sequencing was conducted using an Illumina NovaSeq6000 sequencer. The OmnomicsNGS analysis program was used for data analysis (DNA variant filtering and annotation) (Euformatics, Finland). Reported variants were filtered retaining variations with coding effects, read depths greater than 50, and variants with allelic frequency >10%, with the removal of benign variants according to the ClinVar database. The remaining collection of variations was visually verified in raw data, and probable artifactual variants were eliminated. The list of genes covered by this panel is available at the product website (https://www.illumina.com/products/by-type/clinical-research-products/trusight-oncology-500.html ). In addition, the samples were analyzed using the NGS-based ligation-dependent multiplex RT-PCR assay as described previously [21 ].
4
Transcriptome Analysis of Activated Tregs
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The RNA‐sequencing method has been previously described (Akamatsu et al., 2019 ). Briefly, after in vitro stimulation, Tregs were lysed in RLT buffer (Qiagen) containing 1% 2‐mercaptoethanol, followed by reverse RNA transcription with SMART‐seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech). After cDNA fragmentation by the KAPA Frag kit (KAPA), the sequence libraries were prepared using the KAPA Library IonTorrent (KAPA) preparation kit following the manufacturer's protocol. The cDNA library sequence was performed by IonS5 (Thermo Scientific).
The acquired sequencing results were mapped to the mouse reference genome (mm9; UCSC) using tophat2, and the unmapped sequences were mapped by bowtie2. The combined mapped readings were used to calculate normalized FPKM values using Cuffnorm of Cufflinks package (version 2.2.1, Trapnell Lab) in the default settings.
The acquired sequencing results were mapped to the mouse reference genome (mm9; UCSC) using tophat2, and the unmapped sequences were mapped by bowtie2. The combined mapped readings were used to calculate normalized FPKM values using Cuffnorm of Cufflinks package (version 2.2.1, Trapnell Lab) in the default settings.
5
Mutation Analysis using TruSight Oncology 500
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Mutation analysis was performed using the TruSight Oncology 500 assay (Illumina, San Diego, CA). Total nucleic acid was extracted using the FFPE DNA kit (automated on RSC 48 Instrument, Promega, Madison, Wisconsin, USA). Purified DNA was quantified using the Qubit Broad Range DNA. The quality of DNA was assessed using the FFPE QC kit (Illumina), and DNA samples having Cq<5 were used for further analysis. After the DNA enzymatic fragmentation with KAPA Frag Kit (KAPA Biosystems, Washington, MA), DNA libraries were generated using the TruSight Oncology 500 assay (Illumina), according to the manufacturer’s protocol.
Sequencing was performed using the NextSeq 550 sequencer (Illumina) following the manufacturer’s guidelines. Data analysis (DNA variant filtering and annotation) was performed using the Omnomics Next-generation sequencing (NGS) analysis software (Euformatics, Finland). The custom variant filter was set up including only non-synonymous variants with coding consequences, read depth greater than 50. Benign variants according to the ClinVar database were excluded as well [13 (link)]. The remaining subset of variants was examined visually, and any apparent artefactual variants were excluded.
Sequencing was performed using the NextSeq 550 sequencer (Illumina) following the manufacturer’s guidelines. Data analysis (DNA variant filtering and annotation) was performed using the Omnomics Next-generation sequencing (NGS) analysis software (Euformatics, Finland). The custom variant filter was set up including only non-synonymous variants with coding consequences, read depth greater than 50. Benign variants according to the ClinVar database were excluded as well [13 (link)]. The remaining subset of variants was examined visually, and any apparent artefactual variants were excluded.
6
Quantification of 5-Hydroxymethylcytosine in mESCs
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The mESC genomic DNA was sheared with KAPA Frag Kit (KK8600, Kapa Biosystems, Wilmington, MA, USA) to average 200 base pair and purified using the manufacturer’s protocol (Ring et al., 2017 (link)). Fragmented mESC genomic DNA measuring 500 ng (average 200 bp) was treated with β-GT (New England Biolabs, Ipswich, MA, USA; Catalog #M0357S) in the presence of UDP-6-N3-Glu and labeled with cyclooctyne-biotin. Subsequently, 5hmC-containing fragments were enriched using Streptavidin beads. The enriched fragments were mixed with control DNAs, CPG methylated pUC19, and unmethylated lambda and subjected to denaturation with NaOH and enzymatic deamination using APOBEC enzyme (New England Biolabs, Catalog #E7120S) to transform C/mC to U but not hmC. Then the converted DNA was tagged with Illumina compatible adapter and amplified to an appropriate library concentration using Accel-NGS Methyl-Seq DNA Library Kit (Swift BioSciences, Ann Arbor, MI, USA) combined with NEB Next Multiplex Oligos for Illumina. Libraries were checked for quality and quantified using Agarose Gel Electrophoresis and Qubit3.0 individually.
7
Single Endometrial Gland Target-Gene Sequencing
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The target-gene sequencing of single endometrial glands for 112 genes was performed as described in our previous studies with some modifications15 (link),59 (link),62 (link),63 . Briefly, 112 genes were selected (Supplementary Data) based on WES data for ovarian endometriosis and normal uterine endometrium15 (link), the mutation profiles in endometriosis-related ovarian cancer64 (link) and in endometrial cancer48 (link), and genes involved in DNA repair pathways65 (link),66 (link).
DNA samples were fragmented using a KAPA Frag Kit (KAPA Biosystems). Sequencing libraries were constructed with a NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs). Libraries of up to 96 samples were pooled in equimolar amounts and then hybridized to probes of a SeqCap EZ Prime Choice System (Roche Diagnostics) in a single enrichment reaction. The DNA probe set was selected by using NimbleDesign (Version 3.8) (http://design.nimblegen.com ). The quantity and size distribution of the captured libraries were assessed by a Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and Bioanalyzer 2100 (Agilent Technologies), respectively. The libraries were then sequenced via the Illumina HiSeq 2500, HiSeq 4000 or NovaSeq 6000 platform with 2×100-bp or 2×150-bp paired-end modules (Illumina).
DNA samples were fragmented using a KAPA Frag Kit (KAPA Biosystems). Sequencing libraries were constructed with a NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs). Libraries of up to 96 samples were pooled in equimolar amounts and then hybridized to probes of a SeqCap EZ Prime Choice System (Roche Diagnostics) in a single enrichment reaction. The DNA probe set was selected by using NimbleDesign (Version 3.8) (
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