sequenced at least 40,000×. Importantly, since coverage distribution is not uniform across a sample, it may be necessary to sequence signifi- cantly deeper to achieve coverage of 40,000× at each position of interest.
Implementation in the laboratory As NGS continues to be pushed to its theo-
retical limits, it is necessary to evaluate protocol performance with known standards before implementation into the clinical workflow. Optimizing assay parameters is perhaps most critical when the outcome can affect the down- stream stratification, diagnosis or treatment of patients. Reference standards can potentially play a key role in engineering checkpoints for validation into an NGS workflow, from pre- analytical steps through informatics analysis. Routine validation ensures consistency be- tween reagent lots and personnel and, when a reference standard is used across multiple labo- ratories and workflows, the data may be shared and used to improve platforms, kits, protocols
and assay development. Orthogonally validat- ed, well-characterized human, genomic DNA reference standards offer a renewable resource with which this can be achieved.
References 1. New York State Department of Health.
Updated and revised: Oncology—molecu- lar and cellular tumor markers. “Next gen- eration” sequencing (NGS) guidelines for somatic genetic variant detection; https://
www.wadsworth.org/labcert TestApproval/ forms/NextGenSeq_ONCO_Guidelines.pdf. Accessed Jun 22, 2015, published Mar 2015.
2. Meldrum, C.; Doyle, M.A. et al. Next-gener- ation sequencing for cancer diagnostics: a practical perspective. Clin. Biochem. Rev. 2011, 32(4), 177–95.
3. Quail, M.A.; Smith, M. et al. A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosci- ences and Illumina MiSeq sequencers. BMC Genomics 2012,13, 341.
4. O’Rawe, J.; Jiang, T. et al. Low concor- dance of multiple variant-calling pipe- lines: practical implications for exome and genome sequencing. Genome Med. 2013, 5(3), 28.
5. Armstrong, J.A. Heterogenous DNA sequencing and the lower limits of minor allele frequency sensitivity; https://
cofactorgenomics.com/heterogenous- dna-sequencing-lower-limits-minor-allele- frequency-sensitivity/. Accessed May 4, 2015, published Oct 2014.
Natalie A. LaFranzo, Ph.D., is product man- ager—next-generation sequencing scien- tist, Horizon Discovery Ltd., 7100 Cambridge Research Park, Waterbeach, Cambridge, U.K.; tel.: +44 (0) 1223 655 580; e-mail:
n.lafranzo@
horizondiscovery.com; www.horizondiscovery. com. Horizon’s reference standards are for research use only and not for diagnostic procedures.
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