Massively parallel sequencing was performed on DNA extracted from EBV–B cells from P1, P2, and P3. In brief, the patients’ DNA was sheared with a Covaris S2 Ultrasonicator (Covaris). An adapter-ligated library was prepared with the Paired-End Sample Prep kit V1 (Illumina). Exome capture was performed with the SureSelect Human All Exon kit (Agilent Technologies). Single-end sequencing was performed on a Genome Analyzer IIx (Illumina), generating 72-base reads. As described elsewhere (Byun et al., 2010 (link)), these sequences were aligned with the human genome reference sequence (hg18 build) using BWA aligner. Three open-source packages were used for downstream processing and variant calling (GATK, SAMtools, and Picard Tools). Substitution calls were made with GATK UnifiedGenotyper, whereas indel calls were made with GATK IndelGenotyperV2. All calls with a read coverage ≤4× and a phred-scaled quality of ≤30 were filtered out. All the variants were annotated with the SeattleSeq SNP annotation. Targeted NGS was performed as described elsewhere (Stoddard et al., 2014 (link)).
S2 ultrasonicator
Manufactured by Covaris
Sourced in United States
The Covaris S2 ultrasonicator is a sample preparation instrument designed for controlled, reproducible acoustic disruption of samples. The S2 provides programmable, non-contact acoustic energy to effectively disrupt samples for downstream analysis.
Automatically generated - may contain errors
Lab products found in correlation
Kapa library preparation kit, by Roche (10 mentions)
Bioanalyzer, by Agilent Technologies (10 mentions)
Seqcap ez library, by Roche (6 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (5 mentions)
Paired end sample prep kit v1, by Illumina (4 mentions)
Nextseq 500, by Illumina (4 mentions)
Truchip chromatin shearing reagent kit, by Covaris (4 mentions)
Genome analyzer iix, by Illumina (3 mentions)
Sureselect human all exon kit, by Agilent Technologies (3 mentions)
Qubit system, by Thermo Fisher Scientific (3 mentions)
Hiseq 2000, by Illumina (3 mentions)
Truseq sbs kit v3, by Illumina (3 mentions)
Truseq pe cluster generation kit v3, by Illumina (3 mentions)
Hiseq 3000 sequencing system, by Illumina (3 mentions)
Kapa hyper prep kit, by Roche (3 mentions)
Qubit broad range double stranded dna assay, by Thermo Fisher Scientific (3 mentions)
Hiscansq, by Illumina (2 mentions)
Kapa dna library preparation kit, by Roche (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Dnbseq t7rs sequencer, by MGI Tech (2 mentions)
75 protocols using s2 ultrasonicator
1
Exome Sequencing and Variant Analysis
2
Whole-exome Sequencing and Variant Analysis
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Whole-exome sequencing was performed on both affected patients. Genomic DNA was sheared with a Covaris S2 Ultrasonicator (Covaris). An adaptor-ligated library was prepared with the Paired-End Sample Prep kit V1 (Illumina). Exome capture was performed with the SureSelect Human All Exon v2 kit (Agilent Technologies), covering 38 Megabases (Mbs) of the genome. Single-end sequencing was performed on an Illumina Genome Analyzer IIx. The sequences were aligned with the human genome reference sequence (hg19/GRCh38 build), with BWA-MEM aligner [21 (link)]. Downstream processing was carried out with the Genome Analysis Toolkit (GATK) [22 (link)], SAMtools [23 (link)], and Picard Tools (http://broadinstitute.github.io/picard/ ). Substitution and indel calls were both made with GATK HaplotypeCaller v3.3. All calls with a Phred-scaled quality ≤30 were filtered out. Variant annotation was based on the Human genome assembly GRCh38 as implemented in the Ensembl browser (release 88) as previously described [24 (link)–26 (link)].
3
Comprehensive Cancer Gene Profiling
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Before library construction, genomic DNA extracted from PBL or FFPE specimen was sheared to 300 bp fragments with a Covaris S2 ultrasonicator (Covaris). KAPA Library Preparation Kit (Kapa Biosystems) was adopted to prepare indexed Illumina NGS libraries from PBL DNA, tumor DNA, and plasma DNA. Target enrichment was performed with a custom SeqCap EZ Library (Roche NimbleGen). The capture probe designed based on cancer genomic regions of 59 genes or 1021 genes was used in this study to explore the comprehensive genetic properties of advanced solid tumors. Capture hybridization was carried out according to the manufacturer's protocol. Following hybrid selection, the captured DNA fragments were amplified and then pooled to generate several multiplex libraries.
4
RNA-Seq Library Preparation from Trace RNA
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For mouse and human specimens, RNA extraction was performed using Qiagen miRNeasy kit (cat. 1071023) with on column DNase treatment per the manufacturer’s recommendations. The Clontech Smarter Ultra Low Input RNA kit (Takara Bio, Cat. 634848) was used to generate cDNA from 150 pg total RNA following the manufacturer’s recommendations. Amplified cDNA was purified using SPRI Ampure Beads (Beckman Coulter, Cat. A63880) and the quality and quantity were measured using a High Sensitivity DNA chip on the Agilent 2100 Bioanalyzer (Agilent Technologies). cDNA was sheared to an average length of 300 basepairs using a Covaris S2 ultrasonicator (Covaris) and libraries were generated with the Clontech Low Input Library Prep kit (Takara Bio, Cat. 634947). The samples were uniquely barcoded, pooled, and sequenced on a single lane of the NextSeq 500 (Illumina). A total of 300 million paired-end, 151 base pair reads were obtained, resulting in 50 million reads per sample.
5
Whole Exome Sequencing Library Preparation
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DNA samples were quantified using the Qubit system (Life Technologies). Three µg of DNA was fragmented using the Covaris S2 Ultrasonicator (Covaris, Woburn, MA). The samples were then analyzed on the Bioanalyzer (Agilent Technology, Santa Clara, CA) for correct fragment sizes. Libraries were constructed using the “Agilent SureSelect XT Human All Exon 50Mb library kit” or “Agilent SureSelect_XT_Human_All_Exon_V5” for Illumina Paired End Sequencing (v3 protocol version 1.4.1 or G7530 90000_SureSelect_IlluminaXTMultiplexed_1_5) (Agilent Technologies) according to instructions. The concentration of each library was determined by use of the Qubit and the Bioanalyzer. Whole exome sequencing was performed on the Illumina HiScanSQ with 2x76 bp paired‐end reads using True Seq SBS HS‐v3 clustering and sequencing chemistry (Illumina, San Diego, CA).
6
Targeted Sequencing of PTCL Mutations
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One μg each of genomic DNA sample was sheared into fragments of 300 bp in length by sonication (Covaris S2 ultrasonicator, Covaris, Woburn, MA, USA) before library construction. The KAPA DNA Library Preparation Kit (Kapa Biosystems, Wilmington, MA, USA) was used for the preparation of indexed Illumina next-generation sequencing (NGS) libraries of gDNA. A custom SeqCap EZ Library (Roche NimbleGen, Madison, WI, USA) was applied for target enrichment. To investigate the genetic properties of PTCL, we designed a capture probe basing on genomic regions of approximately 2.4 Mb from 659 genes (see Additional file 1 : Table S1) that are frequently mutated in PTCLs and other common hematological malignancies. Barcoded libraries were hybridized according to the manufacturer’s protocol. DNA fragments captured from the hybrid selection were then amplified and combined to generate various libraries.
7
Chromatin Immunoprecipitation Workflow
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Cells were crosslinked using 1% formaldehyde for 10 min at 298 K. Formaldehyde was diluted to a final concentration of 125 mM by adding 5 M glycine. Nuclear extracts were collected and sonicated to obtain 300 bp chromatin fragments using the Covaris S2 ultrasonicator (Covaris, Woburn, MA). 100 μg of chromatin was incubated with 5.0 μg of AR antibodies (Sigma EMD Millipore, PG21, 06–680, Darmstadt, Germany) overnight at 277 K followed by incubation with 30 μl of protein A/G beads for 4 hours. After four washes, crosslinking was reversed, and chromatin was digested with ribonuclease A (RNaseA) followed by proteinase K. The DNA was purified using spin columns. The size of the DNA was confirmed by a bioanalyzer (Agilent Biotechnologies, Savage, MD).
8
Exome Sequencing and Variant Analysis
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Genomic DNA was isolated by phenol-chloroform extraction from peripheral blood cells or primary fibroblasts from the patient. DNA (3 µg) was sheared with a Covaris S2 Ultrasonicator (Covaris). An adapter-ligated library was prepared with the TruSeq DNA Sample Prep Kit (Illumina). Exome capture was performed with the SureSelect Human All Exon 50 Mb kit (Agilent Technologies). Paired-end sequencing was performed on an Illumina HiSeq 2000 (Illumina), generating 100-base reads. The sequences were aligned with the human genome reference sequence (hg19 build), with the Burrows-Wheeler Aligner. Downstream processing was performed with the Genome Analysis Toolkit (GATK), SAMtools, and Picard Tools (http://picard.sourceforge.net ). Substitution and indel calls were made with the GATK Unified Genotyper and GATK IndelGenotyperV2, respectively. All calls with a Phred-scaled single nucleotide polymorphism quality ≤20 and a read coverage ≤2 were filtered out. All variants were annotated with annotation software developed in-house. For the Sanger sequencing of TLR3 and IFIH1 variants, the exons of TLR3 and IFIH1 were amplified by PCR, purified by ultracentrifugation through Sephadex G-50 Superfine resin (Amersham-Pharmacia-Biotech), and sequenced with the Big Dye Terminator Cycle Sequencing Kit on an ABI Prism 3700 apparatus (Applied Biosystems).
9
Comprehensive Cancer Gene Panel NGS
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DNA was extracted from fresh-frozen tissues or formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens using the QIAamp DNA Mini Kit (Qiagen) and the ReliaPrep FFPE gDNA Miniprep System (Promega). DNA was extracted from plasma specimens using the QIAamp Circulating Nucleic Acid Kit (Qiagen). All samples were obtained from patients after signing informed consent. Prior to pooling, the samples’ concentrations were quantified by Qbit. A minimum of 15 ng of cell-free DNA was required for NGS library construction. Next, the DNA was sheared into fragments at a 200–250 bp peak using a Covaris S2 ultrasonicator (Covaris, Inc.), and indexed NGS libraries were prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB). Finally, the 1,021 panel (Integrated DNA Technologies, Inc.), covering approximately 1.5 Mbp of the genome and targeting 1,021 cancer-related genes, was used for hybridization enrichment. The indexed libraries were sequenced using a 100-bp paired-end configuration on a DNBSEQ-T7RS sequencer (MGI Tech) or Gene+Seq-2000 sequencing system (GenePlus-Suzhou), producing 3 Gb of data for fresh specimens/FFPE.
10
Targeted Sequencing of TP53 and Genes
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DNA from Z138-CytNS, Z138-CytES and Z138-CytR cells were purified using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) and thereafter quantified using the Qubit system (Life Technologies, Carlsbad, CA, USA). Two μg of DNA were fragmented using the Covaris S2 Ultrasonicator (Covaris, Woburn, MA, USA) and DNA fragments from 64 target genes, including TP53 were captured using SureselectXT Custom 3–5.9 Mb library kit (Agilent Technology, Santa Clara, CA, USA). Before capture, eight samples were pooled, and the molarity of the pooled library was determined based on and DNA fragment size distribution measured on a Bioanalyzer (Agilent) and concentration measured by Qubit. Sequencing was performed on the Illumina HiSeq 2500 (Illumina, San Diego, CA, USA) with 2 × 101 bp paired end reads.
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