Bioscentia’s multigene panels use customised sequence capture libraries and multiplexed sequencing.

  • Exons and flanking intronic sequences (35 bp) are targeted and enriched from the patient’s DNA sample using the NimbleGen SeqCap EZ Choice sequence capture approach.

  • DNA samples are then pooled and sequenced in a multiplexing procedure using Illumina MiSeq or HiSeq sequencing-by-synthesis technology. For gene panels, the average coverage is >200-fold, with typical coverage being 400- to 500-fold.
  • Mapping and coverage statistics are generated from the mapping output files using GATK. The high level and reproducibility of coverage enables CNV analysis.
  • The interpretation of the analytical data is managed by Bioscientia’s in-house bioinformatics team using a stepwise filtering process that has been developed and validated by Bioscientia, and published in the peer-reviewed literature.
  • Sequence variants of interest are verified by Sanger sequencing. CNVs are validated by commercial MLPA kit (if available) or by RT-qPCR. If other family members are available, sequence of sequence variants with the disease state will be assessed.

For whole exome sequencing,

  • Exons and flanking intronic sequences (35 bp) are targeted and enriched from the patient’s DNA sample using the NimbleGen SeqCap EZ Choice sequence capture approach.
  • DNA samples are then pooled and sequenced in a multiplexing procedure using Illumina MiSeq or HiSeq and paired-end sequencing (2 x 100 bp) to achieve an average coverage of 100-fold.
  • As with gene panels,
    • statistics are generated using GATK
    • interpretation of variants is managed with Bioscientia’s own bioinformatic pipeline
    • variants of interest are verified by Sanger sequencing, MLPA, or RT-qPCR, and by segregation studies (if possible).

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