For plasma samples, ~1 ng of cfDNA was used as the input for library preparation, and libraries were prepared using a ThruPLEX DNA-seq Library Prep Kit according to the manufacturer’s protocol. For blood samples, libraries were prepared with 200 ng of genomic DNA using a SureSelect Library Preparation Kit (Agilent Technologies) according to the manufacturer’s instructions [21 (link),55 (link)]. For exome enrichment, SureSelect Human All Exon V6 + UTRs probe sets were used. The captured libraries were amplified with 14 cycles of PCR, and the quality and concentration of libraries were assessed using the TapeStation 2200 system (Supplementary Figure S1B ). Libraries were indexed with barcodes to allow sample pooling for multiplexed exome capture and sequencing. An IlluminaNovaSeq 6000 DNA sequencer (Illumina) was used to conduct WES [18 (link)].
Sureselect library preparation kit
Manufactured by Agilent Technologies
The SureSelect library preparation kit is a laboratory equipment product designed for targeted DNA sequencing. It provides a streamlined workflow for sample preparation, allowing users to selectively enrich specific genomic regions of interest prior to sequencing. The kit includes necessary reagents and tools for key steps in the library preparation process.
Automatically generated - may contain errors
Lab products found in correlation
Sureselectxt human all exon v4, by Agilent Technologies (2 mentions)
Herculase 2, by Agilent Technologies (2 mentions)
Agilent sureselectxt humanallexon v4 50 mb, by Agilent Technologies (2 mentions)
Streptavidin coated beads, by Thermo Fisher Scientific (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Qiaamp genomic dna kit, by Qiagen (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
Paired end sample preparation kit, by Illumina (1 mentions)
Hiseq 4000 genome analyzer, by Illumina (1 mentions)
Eland, by Illumina (1 mentions)
Gaiix, by Illumina (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
8 protocols using sureselect library preparation kit
1
Exome Sequencing of Circulating Cell-Free DNA
2
Exome sequencing for variant identification
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Exome sequencing was performed on patient and both parents using the Agilent SureSelectXT HumanAllExon V4 (50 Mb) (Agilent Technologies). Briefly, 3 μg of genomic DNA was sheared in 130 μl of low TE buffer to a peak size of 150–200 bp using Covaris E220, and then purified with AmpPure XP beads to remove fragments less than 100 bp. The purified DNA fragments were then subjected to Agilent SureSelect Library preparation Kit, ILM, to be end-repaired, A-tailed, and ligated to indexing-specific paired-end adaptor. The adapter-ligated libraries were amplified for five cycles using Herculase II (Agilent Technologies). Amplified Pre-capture libraries (750 ng) were concentrated in 3 μl and hybridized to the target specific baits (SureSelectXT Human All Exon V4; Agilent Technologies) according to the manufacturer’s recommendations. Hybridized material was captured using streptavidin-coated beads (Invitrogen, Paisley, UK) and amplified for 10 cycles. Captured libraries were pooled in pairs and paired-end sequenced on one lane of the Illumina HiSeq 2000 at the Stanford Center for Genomics and Personalized Medicine.
3
Exome Sequencing and Variant Analysis
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Genomic DNA extracted from whole blood with the use of QIAamp genomic DNA kit (Qiagen) was applied for exome enrichment by SureSelect library preparation kit (Agilent Technologies) and sequencing on the genome analyser HiSeq 4000 (Illumina), both performed at Macrogen. The data analysis was performed as previously described.13 Candidate variants were confirmed by standard PCR‐Sanger sequencing using in‐house Primers (Table S1 ). All Sanger sequence analyses were performed by Geneious software (Geneious 10.2.2, Biomatters Ltd.).
4
Whole Genome Sequencing of Tumor and Matched Normal Samples
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Genomic libraries with insert sizes of 300bp-600bp were derived from native DNA from 39 tumor and 17 matched normal fresh frozen samples using Illumina® paired end sample preparation kits according to manufacturers instructions. Following cluster generation, 100bp paired-end sequence data was generated using Illumina HiSeqs and was subsequently aligned to the reference human genome (NCBI build37) using BWA. Whole genome libraries from a single FFPE tumor (PD8948c) and matched fresh frozen normal sample (PD8948b) were prepared using Agilent Technologies Sure Select library preparation kit (Custom library kit (cat no. 930075) http://www.agilent.com/search/?Ntt=930075 following manufacturers instructions. 150bp paired end sequence data (with average insert sizes of 319bp and 481bp respectively) was generated using Illumina X10. The average genome wide sequence coverage of tumors and matched normal samples was 42 and 31 fold respectively (Table S2 ).
5
Genetic Analysis of FVII Deficiency
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After obtaining informed consent from all individuals recruited in the study, genomic DNA was isolated from their peripheral blood. The 9 F7 exons, their intronic boundaries, and untranslated regions of F7 gene were polymerase chain reaction (PCR)-amplified, followed by Sanger sequencing with the use of previously published primer sequences [2] (link). Sequence analyses were performed with Geneious Pro (version 10.0.6). Identified genomic variations were reported according to Human Genomic Variation Society nomenclature as presented in EAHAD F7 gene variant database (https://f7-db.eahad.org/ ).
Through PCR-Sanger sequencing, no causative mutation was identified in the entire F7 coding sequence of 3 out of 66 FVII-deficient patients. Whole-exome sequencing (WES) was therefore planned for those 3 cases. Exome enrichment was done by SureSelect library preparation kit (Agilent Technologies) and sequencing was performed on the HiSeq 4000 genome analyzer (Illumina), both at Macrogen The data were then analyzed as previously described [12 (link)]. Briefly, the reads were mapped to the human genome reference sequence (b37) using BWA v0.7.16 and further processed by Genome Analysis ToolKit v4.0.8 [13 (link)] and ANNOVAR [14 (link)]. Our in-house pipeline was used to filter and prioritize the variants.
Through PCR-Sanger sequencing, no causative mutation was identified in the entire F7 coding sequence of 3 out of 66 FVII-deficient patients. Whole-exome sequencing (WES) was therefore planned for those 3 cases. Exome enrichment was done by SureSelect library preparation kit (Agilent Technologies) and sequencing was performed on the HiSeq 4000 genome analyzer (Illumina), both at Macrogen The data were then analyzed as previously described [12 (link)]. Briefly, the reads were mapped to the human genome reference sequence (b37) using BWA v0.7.16 and further processed by Genome Analysis ToolKit v4.0.8 [13 (link)] and ANNOVAR [14 (link)]. Our in-house pipeline was used to filter and prioritize the variants.
6
Exome sequencing for variant identification
7
Exome Sequencing of 107 Samples
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For 107 samples and 20 matched control samples, 50–200 ng DNA was sheared into 300–500-bp fragments using the Covaris ultrasonic system (Covaris). The fragmented DNA was end-repaired, 5′-phosphorylated, and ligated to sequencing adaptors using the Sure Select library preparation kit (Agilent Technologies) following the manufacturer’s instructions. The whole exonic regions of each sample were captured using the Sure Select V6 whole exon kit (Agilent Technologies).
8
mRNA Sequencing for Gene Mutation Screening
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In this study, gene mutations were screened upon mRNA sequencing. The libraries for mRNA sequencing were prepared according to the SureSelect library preparation kit (Agilent Technologies, Santa Clara, CA) and subjected to massively parallel sequencing with a GAIIX or Hiseq2500 sequencer (Illumina, San Diego, CA) using a single-end 36-bp or 50-bp sequencing length protocol. Sequenced tags were aligned to the human reference genome (build hg19) using ELAND (Illumina), and gene expression was normalized to the amount of reads per kilobase of exon per million mapped (rpkm).
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