Webinar: Important Factors in Designing Accurate and Reliable NGS Assays

 

July, 2014
11:00am – 12:00pm (EST)
Presenter: Patricia W. Mueller, Ph.D.

 

 

Researchers, clinical laboratories, and companies are developing next-generation sequencing (NGS) applications that include newborn screening (NBS). Accurate NGS sequence depends on certain key processes. It has recently been determined that in the human genome approximately 17% of the known functional genes have pseudogenes. In order to design accurate and reliable next-generation sequencing assays it is important to check for pseudogenes associated with the genes of interest and design appropriate assays or validation for these genes. Pseudogenes cause a lack of specificity and will result in false reports. For PCR enrichment one should map known mutations and variants and avoid these regions when designing primers. Check primer specificity, and overlap amplicons so that primer regions are sequenced. When using hybrid capture arrays or beads, the oligonucleotides for the genes of interest should be checked for specificity. When sequencing, an average coverage of 30X will result in many regions with little or no coverage. A sequencing run should have adequate coverage to reliably capture the mutations and variants of interest. A quality score of 30, a commonly used quality score cut off in data analysis, means that the probability of an incorrect base call is 1 in 1000. Filtering data with this quality score will result in approximately 1 million incorrect base calls / Gb of NGS data. NGS relies on good coverage to overcome the high error rate. The examination of samples with known mutations and healthy controls allows the determination of the percentage of reads that are needed to accurately call a heterozygote. Finally, it is important to trim primers from the data sequences in addition to adaptors so that the resulting data for these regions is from target regions in samples. Careful attention to these steps in the NGS processes will yield more accurate and reliable data.

 

Panelists:
	Speaker: Patricia W. Mueller, PhD. Chief Molecular Risk Assessment Laboratory (MRAL) Newborn Screening and Molecular Biology Branch Centers for Disease Control and Prevention (CDC)
	Moderator: Dawn Van Dam President and CEO One Million Solutions in Health