Libraries were prepared consistently across three sequencing centers (Annoroad (ARD), NovoGene (NVG) and WuXi NextCODE (WUX)) using 200 ng of DNA with the Illumina TruSeq DNA nano following manufacturer’s instructions. DNA fragmentation was achieved using a Covaris (LE220) instrument with a target size of 350bp. All libraries were assessed for quantity and quality using the Qubit 3.0 fluorometer with the Quant-iT dsDNA HS Assay kit (ThermoFisher Scientific, Q32854) and the Agilent 2100 Bioanalyzer or TapeStation instrument. All materials were prepared with three replicates in a single batch at each sequencing center. They were then sequenced on the Illumina X10 platform with paired end 150 bp read length leveraging synthesis (SBS) chemistry per the manufacturer’s instructions.
Le220 instrument
Manufactured by Covaris
Sourced in United States
The LE220 instrument is a laboratory equipment designed for sample preparation. It utilizes advanced technology for efficient and controlled sample processing. The core function of the LE220 is to provide a consistent and reproducible method for sample preparation.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq x platform, by Illumina (3 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Truseq nano dna, by Illumina (1 mentions)
Quant it dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
X10 platform, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Accel ngs methyl seq dna library preparation kit, by Integrated DNA Technologies (1 mentions)
Qubit dsdna assay, by Thermo Fisher Scientific (1 mentions)
Tapestation, by Agilent Technologies (1 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
Quant it fluorescence assay, by Thermo Fisher Scientific (1 mentions)
Truseq nano dna ht kit, by Illumina (1 mentions)
Hiseq x, by Illumina (1 mentions)
Hiseq x system, by Illumina (1 mentions)
Truseq nano dna library prep kit, by Illumina (1 mentions)
Kapa library quantification kit abi prism, by Illumina (1 mentions)
Bioanalyzer high sensitivity dna chip, by Agilent Technologies (1 mentions)
Truseq dna sample preparation kit, by Illumina (1 mentions)
11 protocols using le220 instrument
1
High-throughput DNA Sequencing Workflow
2
Sensitive DNA Methylation Profiling
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DNA was isolated manually from 500µL serum using the QIAamp DNA Blood Mini kit (Qiagen, Venlo, NL) and 0.03 mg of linear acrylamide (Invitrogen, AM9520). DNA concentration was measured using the Qubit™ dsDNA assay (Thermo Fisher Scientific). Extracted DNA underwent shearing treatment on Covaris LE220 instrument according to the manufacture’s recommendations (Massachusetts, USA) and using 1% trueSHEAR™ buffer (Covaris, Massachusetts, USA), followed by the bisulphite treatment performed using Zymo EZ-96 DNA Methylation-Lightning kit on a 96-well plate (D5033, Zymo Research, USA). We monitored the distribution of DNA size in samples throughout this process on TapeStation (Agilent, USA) according to recommendations from the manufacturer. All 96 samples with isolated DNA were subjected to Accel-NGS® Methyl-Seq DNA library preparation kit (Swift Biosciences, USA) with the adaptase module (Swift Biosciences, USA). The polymerase used in the extension reaction is a uracil-tolerant high-fidelity proofreading polymerase. The nine PCR amplification cycles were performed on 4–20 ng of DNA.
3
Whole-Genome Sequencing of Genomic DNA
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Genomic DNA extracted from tissue was subjected to whole-genome sequencing by Human Longevity Inc. Whole-genome sequencing was performed to a depth of 83× with a distinct coverage of 78×. Briefly, genomic DNA was quantified with a Quant-iT fluorescence assay (Life Technologies) and an eight-point standard curve. The genomic DNA was then normalized and sheared with a Covaris LE220 instrument. Next-generation sequencing library preparation was carried out using the TruSeq Nano DNA HT kit (Illumina), essentially following the manufacturer's recommendations. Individual DNA libraries were characterized in regard to size and concentration using a LabChip DX Touch (PerkinElmer) and Quant-iT (Life Technologies), respectively. Libraries were normalized to 2–3.5 nM and stored at −20°C until used. Normalized DNA libraries were combined into multisample pools and clustered on cBot cluster stations following the manufacturer's recommendations. Flow cells were subsequently sequenced on the HiSeqX (Illumina) utilizing a 150 base paired-end single index format. Analysis of structural variants was then performed by Personal Genome Diagnostics using methods previously described in detail (Sausen et al. 2013 (link); Jones et al. 2015 ).
4
Whole Genome Sequencing of Participant Cohort
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WGS of index participants was performed with the Illumina (San Diego, CA) HiSeq X system at The Centre for Applied Genomics in Toronto, Ontario, Canada from DNA extracted from whole blood. DNA was quantified using the Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA) High Sensitivity Assay, and sample purity was checked using the Nanodrop (Thermo Fisher Scientific, Waltham, MA) OD 260/280 ratio. Following the manufacturer’s recommended protocol, 100 ng of DNA were used as input material for library preparation using the Illumina TruSeq Nano DNA Library Prep Kit. In brief, DNA was fragmented to an average of 350 base pairs by sonication on a Covaris (Woburn, MA) LE220 instrument. Fragmented DNA was end-repaired and A-tailed and indexed TruSeq Illumina adapters added by ligation before library amplification. Libraries were assessed using Bioanalyzer DNA High Sensitivity chips (Agilent Technologies, Santa Clara, CA) and quantified by quantitative polymerase chain reaction using the Kapa Library Quantification Illumina/ABI Prism Kit protocol (KAPA Biosystems, Roche, Basel, Switzerland). Validated libraries were pooled in equimolar quantities and paired-end sequenced on an Illumina HiSeq X platform, following Illumina’s recommended protocol, to generate paired-end reads of 150 bases in length.
5
Exome Sequencing of Brain and Blood
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A Covaris LE220 instrument was used to fragment 3 μg of genomic DNA (from brain and blood) to an average size of 200 bp. Short insert libraries were obtained using the Illumina TruSeq DNA Sample Preparation Kit. Exonic sequences were enriched using NimbleGen Sequence Capture Human Exome 2.1M Array. Paired-end sequences of 91 nucleotides from each end were generated using an Illumina HiSeq 2000 instrument to an average of 50x coverage. Sequences were generated in FastaQ format.
6
Agilent Exome Sequencing Protocol
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Exome libraries were prepared using Agilent SureSelect Human Exome Library Preparation V5 kit and the Agilent Bravo Automation System followed by paired-end sequencing on an Illumina HiSeq 2500 platform. Genomic DNA (750 ng) was fragmented to 200-bp on average using a Covaris LE220 instrument. Sheared DNA was endrepaired and the 3 0 ends adenylated prior to ligation of adapters with overhang-T. Genomic library was amplified by PCR using 10 cycles and hybridized with biotinylated probes that target exonic regions; the enriched exome libraries were amplified by an additional 8 cycles of PCR. Exomic libraries were validated on a Bioanalyzer 2100 DNA High Sensitivity chip (Agilent Technologies) for size and by qPCR using the Kapa Library Quantification Illumina/ABI Prism Kit protocol (KAPA Biosystems) for quantities. Exome libraries were pooled and sequenced with the TruSeq SBS sequencing chemistry using a V4 high throughput flowcell on a HiSeq 2500 platform following Illumina's recommended protocol. Approximately 6 to 8 gigabases of raw paired end data of 126-bases were generated per exome library.
7
Optimized 3C Library Preparation
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3C libraries were prepared from sorted populations from the thymus and spleen/lymph nodes as described previously24 (link) with the following changes: DpnII enzymes were used for restriction digest, with a total of 500 U fresh restriction enzyme added every 5–7 h, for a total incubation of 16–24 h after initial addition of restriction enzyme. After digest, DpnII was heat inactivated and a ligation reaction with 200 U of T4 DNA ligase (NEB) was set up overnight at room temperature with gentle rotation. Two aliquots of 5 µg of 3C library was then sonicated to 200 bp using a Covaris LE220 instrument (190 s shearing time; 30% duty factor; 450PIP, 200 cycles per burst). We indexed libraries with Illumina Truseq indexed sequencing adaptors using NEBnext reagents (E6000, E6040, E7335, or E7500) for end repair, dA labeling, adaptor ligation, and PCR indexing, primarily following the manufacturer’s instructions. To maximize library complexity, we used 10 μg of input material split into two reactions and pooled them after indexing. We assessed libraries using an Agilent Bioanalyser or D1000 Tapestation after addition of sequencing adaptors.
8
Mouse Tail DNA Extraction and Exome Sequencing
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DNA was extracted from mouse tails using standard methods (Supplemental Methods ). For sequencing by The Centre for Applied Genomics (TCAG), 500 ng of genomic DNA was fragmented to 200 bp on average using a Covaris LE220 instrument. Sheared DNA was end-repaired and the 3′ ends adenylated before ligation of adapters with overhang-T. PCR amplified genomic libraries were exome captured, pooled, and sequenced with the TruSeq SBS sequencing chemistry using a V4 high-throughput flowcell on a HiSeq 2500 platform following Illumina's recommended protocol. Approximately 6–8 gb of raw paired end data of 126 bases were generated per exome library. Inbred strain polymorphisms as well as systematic sequencing artifacts, were removed from consideration if identified in the parental strains, dbSNP, or other founder males sequenced in this study, and detrimental variants were called using standard tools and custom scripts (Supplemental Code ) (Li et al. 2009 (link); Li and Durbin 2010 (link)). Candidate lesions were confirmed by Sanger sequencing before genotyping N3 animals (Supplemental Fig. S3 ). Primer sequences for each locus are in Supplemental Table S4 .
9
Whole Genome Sequencing of Tumor and Normal Tissue
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For each of 53 patients, two samples of 1μg genomic DNA from tumor and histologically normal axillary lymph nodes were whole genome sequenced at The McDonnell Genome Institute at Washington University (St Louis MO, USA) on an Illumina HiSeqX platform. Briefly, manual dual indexed libraries were constructed with 1μg of FFPE genomic DNA for the 53 tumor/normal pairs using the Accel-NGS 2S Plus Library Kit (Swift, MI, USA). Samples were fragmented on the Covaris LE220 instrument with 350bp target insert size. PCR cycle optimization was performed to prevent over-amplification of the libraries. The concentration of each library was determined through qPCR (Kapa Biosystems, MA, USA). For the normal samples, each library was loaded on one lane of a HiSeqX flow cell, whereas for tumor samples, each library was loaded across two lanes of a HiSeqX flow cell. 2×150 paired-end sequence data were generated at a target depth of 30×(normal) and 60(tumor) haploid genome coverage. All sequencing data are available in EGA under accession “EGAS00001002685”. Further details are provided in the Supplementary Methods.
10
Whole-Genome Sequencing for Variant Identification
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Subjects that were identified with DNAm profiles similar to the WS profile were subjected to next-generation sequencing for variant identification. About 1 μg of genomic DNA was submitted to TCAG for genomic library preparation and whole-genome sequencing. DNA samples were quantified using Qubit High Sensitivity Assay and sample purity was checked using Nanodrop OD260/280 ratio. 700 ng of DNA was used as input material for library preparation using the Illumina TruSeq PCR-free DNA Library Prep Kit following the manufacturer’s recommended protocol. In brief, DNA was fragmented to an average size of 400 bp using sonication on a Covaris LE220 instrument; fragmented DNA was end-repaired, A-tailed, and indexed TruSeq Illumina adapters with overhang-Ts added to the DNA. Libraries were validated on a Bioanalyzer DNA High-Sensitivity chip to check for size and absence of primer dimers and quantified by qPCR using the Kapa Library Quantification Illumina/ABI Prism Kit protocol (KAPA Biosystems). The Validated library was paired-end sequenced on the Illumina HiSeq X platform following Illumina’s recommended protocol to generate paired-end reads of 150-bases in length.
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