Library construction and exome capture have been automated (Perkin-Elmer Janus II) in 96-well plate format. 1 μg of genomic DNA was subjected to a series of shotgun library construction steps, including fragmentation through acoustic sonication (Covaris), end-polishing and A-tailing, ligation of sequencing adaptors and PCR amplification with dual 8 bp barcodes for multiplexing. Libraries underwent exome capture using the Roche/Nimblegen SeqCap EZ v2.0 (~36.5 MB target). Prior to sequencing, the library concentration was determined by fluorometric assay and molecular weight distributions verified on the Agilent Bioanalyzer (consistently 150 ± 15 bp).
Janus 2
Manufactured by PerkinElmer
The Janus II is a liquid handling workstation designed for automated sample preparation and assay workflows. It features a modular design, precise liquid handling, and integrated plate management capabilities to streamline laboratory processes.
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6 protocols using janus 2
1
Automated Exome Capture Library Construction
2
Automated Exome Capture Library Construction
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Library construction and exome capture have been automated (Perkin-Elmer Janus II) in 96-well plate format. 1 μg of genomic DNA was subjected to a series of shotgun library construction steps, including fragmentation through acoustic sonication (Covaris), end-polishing and A-tailing, ligation of sequencing adaptors and PCR amplification with dual 8 bp barcodes for multiplexing. Libraries underwent exome capture using the Roche/Nimblegen SeqCap EZ v2.0 (~36.5 MB target). Prior to sequencing, the library concentration was determined by fluorometric assay and molecular weight distributions verified on the Agilent Bioanalyzer (consistently 150 ± 15 bp).
3
Whole Exome Sequencing Protocol
4
Automated High-Throughput DNA Sequencing
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Genomic DNA was extracted from whole blood or buffy coats using Qiagen kits (Qiagen, Chatsworth, CA, USA) or salting out procedures described previously [20 (link),21 (link)]. 1 ug of genomic DNA was sent to the Core lab at Northwest Genomics Center at the University of Washington for sequencing. The quality and integrity of DNA was checked at the Core lab using Agilent’s Analyzer and Tape Station reagents before target capture and library preparation. Library construction and custom capture have been automated (Perkin-Elmer Janus II) in a 96-well plate format. The purified DNA was subjected to a series of shotgun library construction steps, including fragmentation through acoustic sonication (Covaris), end-polishing and A-tailing, ligation of sequencing adaptors, and PCR amplification with 8 bp barcodes for multiplexing. Libraries undergo capture using the Roche/Nimblegen SeqCap EZ custom designed probe. Prior to sequencing, the library concentration was determined by triplicate qPCR and molecular weight distributions verified on the Agilent Bioanalyzer. Barcoded libraries were pooled using liquid handling robotics prior to clustering (Illumina cBot) and loading. Massively parallel sequencing-by-synthesis with fluorescently labeled, reversibly terminating nucleotides was carried out on the HiSeq sequencer.
5
Whole Exome Sequencing Protocol
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DNA samples were submitted to the University of Washington Center for Mendelian Genomics (UW-CMG) where library construction, WES and analysis were performed. Briefly, sequencing libraries were generated for each DNA sample in an automated, 96-well plate format (Perkin-Elmer Janus II). Sample libraries were constructed from 1 μg of genomic DNA which underwent a series of shotgun library construction steps including: acoustic sonication (Covaris), end-repair, A-tailing ligation of unique sequencing adaptors, and PCR amplification. Sample libraries were hybridized to the Nimblegen SeqCap EZ v2.0 target (∼36.5 Mb) in multiplex for a period of 72 hrs. Captured DNA was then purified, PCR amplified, and normalized for sequencing.
Captured DNA was sequenced on Illumina HiSeq machines using paired-end sequencing. Raw sequence data in FASTQ format were aligned to the human genome reference hg19 using the BWA algorithm for the generation of BAM files9 (link). The quality of each sample was assessed for coverage (80% of sequenced target with ≥20× coverage and 90% of target with ≥8× coverage) and Ts/Tv ratios. Additionally, samples were quality controlled to confirm sex using PLINK (v1.90b2m) software, and estimations of kinship were corroborated with pedigrees using KINGv1.4.0 software.
Captured DNA was sequenced on Illumina HiSeq machines using paired-end sequencing. Raw sequence data in FASTQ format were aligned to the human genome reference hg19 using the BWA algorithm for the generation of BAM files9 (link). The quality of each sample was assessed for coverage (80% of sequenced target with ≥20× coverage and 90% of target with ≥8× coverage) and Ts/Tv ratios. Additionally, samples were quality controlled to confirm sex using PLINK (v1.90b2m) software, and estimations of kinship were corroborated with pedigrees using KINGv1.4.0 software.
6
Automated Exome Sequencing Pipeline
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