Fresh frozen samples (500 ng) were subjected to target fragment enrichment using a Twist Library Preparation EF Kit (Twist Bioscience, South San Francisco, CA, USA). Massively parallel sequencing of the isolated fragments was performed using the paired-end option on a NovaSeq 6000 platform (Illumina, San Diego, CA, USA). Paired-end WES reads with nucleotides masked with a quality score <20 were aligned to a merged reference sequence of the human reference genome (GRCh38) and Mus musculus reference genome (mm10) using BWA-MEM. Somatic mutations were called using the Genome Analysis Toolkit (https://gatk.broadinstitute.org/hc/en-us ), MuTect2, VarScan2 (http://varscan.sourceforge.net ), and our in-house somatic caller. Mutations were discarded if any of the following criteria were met: read depth <20, variant allele frequency <0.05, mutation occurring in only one strand of the genome, mutant read number in germline control samples >2, or the variant was present in normal human genomes in either the 1000 Genomes Project dataset (https://www.internationalgenome.org/ ) or our in-house database. Gene mutations were annotated using SnpEff (http://snpeff.sourceforge.net ).
Twist library preparation ef kit
Manufactured by Twist Bioscience
Sourced in United States
The Twist Library Preparation EF Kit is a laboratory equipment product developed by Twist Bioscience. It is designed to facilitate the preparation of DNA libraries for various sequencing applications. The core function of the kit is to enable the efficient and reliable construction of sequencing-ready libraries from DNA samples.
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Lab products found in correlation
Novaseq 6000, by Illumina (4 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Nebnext ultra 2 non directional rna second strand synthesis module, by New England Biolabs (1 mentions)
Protoscript 2 first strand cdna synthesis kit, by New England Biolabs (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Clinical research exome v2, by Agilent Technologies (1 mentions)
Twist human core exome kit, by Twist Bioscience (1 mentions)
Accel ngs 2s hyb dna library kit, by Integrated DNA Technologies (1 mentions)
5 protocols using twist library preparation ef kit
1
Whole Exome Sequencing of Tumor Samples
2
Twist Library Preparation for NGS
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Details on NGS library preparation are provided in the Supplemental Materials and Methods . Briefly, approximately 300 ng of gDNA was used to prepare sequencing libraries using a Twist Library Preparation EF Kit (Twist Bioscience, San Francisco, CA, USA). Target enrichment was performed using a capture probe, and sequencing was conducted on a NovaSeq 6000 instrument (Illumina, San Diego, CA, USA), achieving approximately 150 million reads per sample. Sequencing was performed using a 151-bp, dual-indexed, paired-end sequencing configuration.
3
Transcriptome Profiling by Targeted RNA Sequencing
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Total RNA was extracted from fresh frozen samples using the RNeasy Mini Kit (QIAGEN, Hilden, Germany). Then, 200 ng of RNA was converted to cDNA using the ProtoScript II First Strand cDNA Synthesis Kit and NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs, Ipswich, MA, USA) and subjected to subsequent target enrichment using TOP-RNA-V6 probes with the Twist Library Preparation EF Kit (Twist Bioscience, South San Francisco, CA, USA). Using a paired-end option, massive parallel sequencing of the isolated fragments was conducted using a NovaSeq 6000 (Illumina). Paired-end reads containing masked nucleotides with a quality score < 20 were aligned to the human reference genome (hg38) using STAR (v2.5.2a; https://github.com/alexdobin/STAR ). Mutations were called using an in-house mutation caller based on the SAMtools’ mpileup results. Mutations were discarded if any of the following criteria were met: read depth < 20, variant allele frequency < 0.001, and the presence of the variant in normal human genomes in either the 1000 Genomes Project dataset (https://www.internationalgenome.org/ ) or our in-house database. Gene mutations were annotated using SnpEff (http://snpeff.sourceforge.net ).
4
Targeted cfDNA Next-Generation Sequencing
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NGS libraries were prepared with 20 ng of cfDNA using the Twist Library Preparation EF Kit (Twist Bioscience, San Francisco, USA) according to manufacturer’s instructions with minor modifications. The fragmentation time at 32°C was skipped followed by an enzyme inactivation at 65°C for 30 minutes. For adapter ligation and barcoding, xGen Duplex CS Adapters and customized IDT Dual-Index Primer (both from Integrated DNA Technologies, cat. no. 1080799) were used. Target enrichment was performed with a custom design enrichment panel containing selected hotspots in ALK, BRAF, EGFR, ERBB2, H3F3A, HIST1H3B, IDH1, IDH2, KIT, KRAS, MET, NRAS, PDGFRA, PIK3CA, ROS1, SRY, TERT, and TP53 (7kb in total). Quality and quantity of cfDNA and final libraries were assessed using the High Sensitivity NGS Fragment Analysis Kit (AATI, Santa Clara, USA, cat. no. DNF-474) and Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, USA, cat. no. Q32854), respectively. Paired-end sequencing was performed on a NovaSeq6000 instrument (Illumina, San Diego, CA) with 2x100 base pairs (bp) read length, yielding at least 8 Gbp per sample (8.1–13.9, mean 11.8).
5
Hyb-based Library Preparation and Exome Capture
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Library preparation was performed as previously described [28 ] using either the Accel-NGS 2S Hyb DNA Library Kit (SWIFT Biosciences, San Francisco, CA, USA) or Twist Library Preparation EF Kit (TWIST Biosciences, San Francisco, CA, USA) [28 ]. For exome capture, either the Twist Human Core Exome Kit (TWIST Biosciences, San Francisco, CA, USA) or Clinical Research Exome V2 (Agilent, Santa Clara, CA, USA) were used and further sequenced by Illumina paired-end sequencing producing a minimum of 26 Gb and 18 Gb of raw sequence data for tumor DNA and normal DNA samples, respectively.
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