Genomic DNA was isolated as indicated in module 1b33 (link) and submitted to the Genewiz company (≥1.5ug DNA per tumor at ≥20 ng/μL; A260/A280 = 1.8–2.0 and RNA-free) for library preparation and sequencing. The following quality control checks were performed for all samples before sequencing: (i) genomic DNA integrity assessment by gel electrophoresis and quantification of total concentration by Qubit assay; (ii) sonication efficiency and library size assessment by capillary electrophoresis using the 5300 Fragment Analyzer system (Agilent); (iii) quantification of library concentration by Qubit assay. Whole-exome and low-pass whole-genome sequencing libraries were generated using the SureSelect Human All Exon v7 kit (Agilent) and SureSelectQXT Library Prep (Agilent), respectively, following the manufacturer’s instructions. Paired-end 150 base-pair (2 × 150) sequencing of the libraries was performed using an Illumina NovaSeq 6000 sequencer.
Sureselectqxt library prep
Manufactured by Agilent Technologies
The SureSelectQXT Library Prep is a laboratory equipment product offered by Agilent Technologies. It is designed for library preparation, a critical step in next-generation sequencing workflows. The core function of this product is to enable the efficient and accurate construction of sequencing libraries from DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
5300 fragment analyzer system, by Agilent Technologies (1 mentions)
Sureselect human all exon v7 kit, by Agilent Technologies (1 mentions)
Novaseq 6000 sequencer, by Illumina (1 mentions)
4200 tapestation system, by Agilent Technologies (1 mentions)
Suredesign, by Agilent Technologies (1 mentions)
Nextseq 500, by Illumina (1 mentions)
2 protocols using sureselectqxt library prep
1
Whole-Exome and Low-Pass Whole-Genome Sequencing
2
Custom Target Enrichment Library Design
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A custom target enrichment library design was performed using the SureDesign (Agilent Technologies, Santa Clara, CA, USA) web application. RNA probes were manufactured to capture a panel of 40 genes selected according to the Spanish Group of MDS (GESMD) guidelines [18 (link)] (total probe size: 213,846 kb; Table S1 ). This custom design was combined with the “OneSeq 1 Mb CNV Backbone” commercial design (Agilent Technologies) to enable the detection of CNAs and cnLOH with resolutions of 1 and 10 Mb, respectively (total probe size: 2.674 Mb). Sequencing libraries were prepared using the “SureSelectQXT Library Prep” protocol (Agilent Technologies) starting from 50 ng of DNA and a unique 90-minute capture step. The quality and concentration of DNA samples and libraries were assessed with the 4200 TapeStation System (Agilent Technologies), and 10 sample pools were sequenced (75 × 2 cycles) on a NextSeq 500 (Illumina, San Diego, CA, USA). The mean number of reads in .bam files passing the mapping quality filters per sample was 14,226,190 ± 2,825,217 and had a 319 ± 133x average depth (Table S2 ). Ten (17%) samples were randomly selected to perform a 50 and 75% subsampling of available reads (https://github.com/lh3/seqtk , accessed on 10 April 2021) for further validation of the strategy.
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