Whole Exome Sequencing Library Preparation

For GLD1, sequencing libraries were made following the protocol from Roche/Nimblegen’s SeqCap EZ Exome Library v2.0 kit (Roche NimbleGen, Inc). The libraries were then sequenced on a HiSeq2000 (Illumina), with paired-end, 101bp reads. For GLD2 and GLD3, whole exome capture was performed using the SureSelect Target Enrichment System (Agilent). This was followed by sequencing on a HiSeq2000 (Illumina) with 100bp paired end reads. Sequence reads for all families were aligned to the reference genome (hg19) using Novoalign (Novocraft Technologies SdnBhd). Duplicate reads, resulting from PCR clonality or optical duplicates, and reads mapping to multiple locations were excluded from downstream analysis. Depth and breadth of sequence coverage were calculated with custom scripts and the BedTools package^{26 (link)}.

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Fotiou E., Martin-Almedina S., Simpson M.A., Lin S., Gordon K., Brice G., Atton G., Jeffery I., Rees D.C., Mignot C., Vogt J., Homfray T., Snyder M.P., Rockson S.G., Jeffery S., Mortimer P.S., Mansour S, & Ostergaard P. (2015). Novel mutations in PIEZO1 cause an autosomal recessive generalized lymphatic dysplasia with non-immune hydrops fetalis. Nature Communications, 6, 8085.

Publication 2015

SeqCap EZ Exome Library v2.0 kit HiSeq2000 SureSelect Target Enrichment System

Exome Genome Library Optical

Corresponding Organization :

Other organizations : St George's, University of London, Guy's Hospital, King's College London, Stanford University, St George’s University Hospitals NHS Foundation Trust, South West London and St George's Mental Health NHS Trust, King's College Hospital, Centre d'Épidémiologie sur les Causes Médicales de Décès, Assistance Publique – Hôpitaux de Paris, Birmingham Women's Hospital

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Variable analysis

independent variables

Sequencing protocol (Roche/Nimblegen's SeqCap EZ Exome Library v2.0 kit for GLD1, SureSelect Target Enrichment System for GLD2 and GLD3)
Sequencing platform (HiSeq2000, Illumina)

dependent variables

Depth and breadth of sequence coverage

control variables

Alignment to reference genome (hg19) using Novoalign
Exclusion of duplicate reads and reads mapping to multiple locations

controls

No positive or negative controls were explicitly mentioned in the input protocol.

Annotations

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