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Sci Rep. 2017 Jun 13;7(1):3356. doi: 10.1038/s41598-017-03448-8.

Targeted sequencing of both DNA strands barcoded and captured individually by RNA probes to identify genome-wide ultra-rare mutations.

Wang Q1,2,3, Wang X4, Tang PS5,4, O'leary GM5,4, Zhang M6,7,8.

Author information

1
MyOmicsDx Inc., 600 Washington Avenue Suite 100-W, Towson, Maryland, 21204, USA. qwang@myomicsdx.com.
2
MyOmicsDx (Beijing) Inc., North Yong Chang Road Building # 3-3, Economic-Technological Development Area, DaXing, Beijing, 100176, P.R. China. qwang@myomicsdx.com.
3
Clinical Proteomics Technologies Inc., 8101 Sandy Spring Road, Laurel, Maryland, 20707, USA. qwang@myomicsdx.com.
4
MyOmicsDx (Beijing) Inc., North Yong Chang Road Building # 3-3, Economic-Technological Development Area, DaXing, Beijing, 100176, P.R. China.
5
MyOmicsDx Inc., 600 Washington Avenue Suite 100-W, Towson, Maryland, 21204, USA.
6
MyOmicsDx Inc., 600 Washington Avenue Suite 100-W, Towson, Maryland, 21204, USA. mzhang@myomicsdx.com.
7
MyOmicsDx (Beijing) Inc., North Yong Chang Road Building # 3-3, Economic-Technological Development Area, DaXing, Beijing, 100176, P.R. China. mzhang@myomicsdx.com.
8
Clinical Proteomics Technologies Inc., 8101 Sandy Spring Road, Laurel, Maryland, 20707, USA. mzhang@myomicsdx.com.

Abstract

Next Generation Sequencing (NGS) has been widely implemented in biological research and has made a profound impact on patient care. One of the essential NGS applications is to identify disease-causing sequence variants, where high coverage and accuracy are needed. Here, we reported a novel NGS pipeline, termed a Sequencing System of Digitalized Barcode Encrypted Single-stranded Library from Extremely Low (quality and quantity) DNA Input with Probe-based DNA Enrichment by RNA probes targeting DNA duplex (DEEPER-Seq). This method combines an ultra-sensitive single-stranded library construction with barcoding error correction, termed DEEPER-Library; and a DNA capture approach using RNA probes targeting both DNA strands, termed DEEPER-Capture. DEEPER-Seq can create NGS libraries from as little as 20 pg DNA with PCR error correcting capabilities, and capture target sequences at an average ratio of 29.2% by targeting both DNA strands simultaneously with an over 98.6% coverage. Our method tags and sequences each of the two strands of a DNA duplex independently and only scores mutations that are found at the same position in both strands, which allows us to identify mutations with allelic fractions down to 0.03% in a whole exome sequencing (WES) study with a background error rate of one artificial error per 4.8 × 109 nucleotides.

PMID:
28611392
PMCID:
PMC5469810
DOI:
10.1038/s41598-017-03448-8
[Indexed for MEDLINE]
Free PMC Article

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