Development of a robust DNA quality and quantity assessment qPCR assay for targeted next-generation sequencing library preparation

Dang,Jennifer; Mendez,Pedro; Lee,Sharon; Kim,James W.; Yoon,Jun-Hee; Kim,Thomas W.; Sailey,Charles J.; Jablons,David M.; Kim,Il-Jin

doi:10.3892/ijo.2016.3654

Development of a robust DNA quality and quantity assessment qPCR assay for targeted next-generation sequencing library preparation

Authors:
- Jennifer Dang
- Pedro Mendez
- Sharon Lee
- James W. Kim
- Jun-Hee Yoon
- Thomas W. Kim
- Charles J. Sailey
- David M. Jablons
- Il-Jin Kim
View Affiliations

Affiliations: CureSeq Inc., Brisbane, CA 94005, USA, Thoracic Oncology Laboratory, Department of Surgery, University of California San Francisco, San Francisco, CA 94115, USA
Published online on: August 11, 2016 https://doi.org/10.3892/ijo.2016.3654
Pages: 1755-1765
Copyright: © Dang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Next-generation sequencing (NGS) is becoming a standard for genetic analyses of clinical samples. DNAs retrieved from formalin-fixed, paraffin-embedded (FFPE) tissue specimens are commonly degraded, and specimens such as core biopsies are sometimes too small to obtain enough DNA for NGS applications. Thus, it is important to measure both the DNA quantity and quality accurately from clinical samples. However, there is no standard method for DNA quantity and quality analyses for NGS library preparation. We tested four different methods (PicoGreen, Qubit® fluorometry, TaqMan and SYBR-Green-based qPCR assay) and compared each to RNase P TaqMan as a reference control. We found that SYBR-Green-based qPCR assay provides a consistent and accurate DNA quantification while keeping its cost relatively low and the throughput high. We designed a dual-probe SYBR-Green qPCR assay for DNA quantity and quality assessment for targeted NGS library preparation. This assay provides a Dscore (degradation score) of the interrogated DNA by analyzing two different sizes of amplicons. We show an example of a clinical sample with a very high Dscore (high degradation). With a regular DNA quantification, without considering the degradation status, no correct NGS libraries were obtained. However, after optimizing the library condition by considering its poor DNA quality, a reasonably good library and sequencing results were obtained. In summary, we developed and presented a new DNA quantity and quality analysis qPCR assay for the targeted NGS library preparation. This assay may be mostly efficient for the clinical samples with high degradation and poor DNA quality.

View Figures

View References

1	Bennett ST, Barnes C, Cox A, Davies L and Brown C: Toward the 1,000 dollars human genome. Pharmacogenomics. 6:373–382. 2005. View Article : Google Scholar : PubMed/NCBI
2	Hillier LW, Marth GT, Quinlan AR, Dooling D, Fewell G, Barnett D, Fox P, Glasscock JI, Hickenbotham M, Huang W, et al: Whole-genome sequencing and variant discovery in C. elegans. Nat Methods. 5:183–188. 2008. View Article : Google Scholar : PubMed/NCBI
3	Parsons DW, Jones S, Zhang X, Lin JC, Leary RJ, Angenendt P, Mankoo P, Carter H, Siu IM, Gallia GL, et al: An integrated genomic analysis of human glioblastoma multiforme. Science. 321:1807–1812. 2008. View Article : Google Scholar : PubMed/NCBI
4	Shah SP, Morin RD, Khattra J, Prentice L, Pugh T, Burleigh A, Delaney A, Gelmon K, Guliany R, Senz J, et al: Mutational evolution in a lobular breast tumour profiled at single nucleotide resolution. Nature. 461:809–813. 2009. View Article : Google Scholar : PubMed/NCBI
5	Fang LT, Lee S, Choi H, Kim HK, Jew G, Kang HC, Chen L, Jablons D and Kim IJ: Comprehensive genomic analyses of a metastatic colon cancer to the lung by whole exome sequencing and gene expression analysis. Int J Oncol. 44:211–221. 2014.
6	Kang HC, Kim HK, Lee S, Mendez P, Kim JW, Woodard G, Yoon JH, Jen KY, Fang LT, Jones K, et al: Whole exome and targeted deep sequencing identify genome-wide allelic loss and frequent SETDB1 mutations in malignant pleural mesotheliomas. Oncotarget. 7:8321–8331. 2016.PubMed/NCBI
7	Mendez P, Dang J, Kim JW, Lee S, Yoon JH, Kim T, Sailey CJ, Jablons DM and Kim IJ: Comprehensive evaluation and validation of targeted next-generation sequencing performance in two clinical laboratories. Int J Oncol. 49:235–242. 2016.PubMed/NCBI
8	Hadd AG, Houghton J, Choudhary A, Sah S, Chen L, Marko AC, Sanford T, Buddavarapu K, Krosting J, Garmire L, et al: Targeted, high-depth, next-generation sequencing of cancer genes in formalin-fixed, paraffin-embedded and fine-needle aspiration tumor specimens. J Mol Diagn. 15:234–247. 2013. View Article : Google Scholar : PubMed/NCBI
9	Han SW, Kim HP, Shin JY, Jeong EG, Lee WC, Lee KH, Won JK, Kim TY, Oh DY, Im SA, et al: Targeted sequencing of cancer-related genes in colorectal cancer using next-generation sequencing. PLoS One. 8:e642712013. View Article : Google Scholar : PubMed/NCBI
10	Cancer Genome Atlas Research Network. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature. 455:1061–1068. 2008. View Article : Google Scholar : PubMed/NCBI
11	Kandoth C, McLellan MD, Vandin F, Ye K, Niu B, Lu C, Xie M, Zhang Q, McMichael JF, Wyczalkowski MA, et al: Mutational landscape and significance across 12 major cancer types. Nature. 502:333–339. 2013. View Article : Google Scholar : PubMed/NCBI
12	Brennan CW, Verhaak RG, McKenna A, Campos B, Noushmehr H, Salama SR, Zheng S, Chakravarty D, Sanborn JZ, Berman SH, et al; TCGA Research Network. The somatic genomic landscape of glioblastoma. Cell. 155:462–477. 2013. View Article : Google Scholar : PubMed/NCBI
13	Bass AJ, Thorsson V, Shmulevich I, Reynolds SM, Miller M, Bernard B, Hinoue T, Laird PW, Curtis C, Shen H, et al; Cancer Genome Atlas Research Network. Comprehensive molecular characterization of gastric adenocarcinoma. Nature. 513:202–209. 2014. View Article : Google Scholar :
14	Imielinski M, Berger AH, Hammerman PS, Hernandez B, Pugh TJ, Hodis E, Cho J, Suh J, Capelletti M, Sivachenko A, et al: Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing. Cell. 150:1107–1120. 2012. View Article : Google Scholar : PubMed/NCBI
15	Ng SB, Turner EH, Robertson PD, Flygare SD, Bigham AW, Lee C, Shaffer T, Wong M, Bhattacharjee A, Eichler EE, et al: Targeted capture and massively parallel sequencing of 12 human exomes. Nature. 461:272–276. 2009. View Article : Google Scholar : PubMed/NCBI
16	Choi M, Scholl UI, Ji W, Liu T, Tikhonova IR, Zumbo P, Nayir A, Bakkaloğlu A, Ozen S, Sanjad S, et al: Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proc Natl Acad Sci USA. 106:19096–19101. 2009. View Article : Google Scholar : PubMed/NCBI
17	Ng SB, Buckingham KJ, Lee C, Bigham AW, Tabor HK, Dent KM, Huff CD, Shannon PT, Jabs EW, Nickerson DA, et al: Exome sequencing identifies the cause of a mendelian disorder. Nat Genet. 42:30–35. 2010. View Article : Google Scholar :
18	Gawad C, Koh W and Quake SR: Single-cell genome sequencing: Current state of the science. Nat Rev Genet. 17:175–188. 2016. View Article : Google Scholar : PubMed/NCBI
19	de Bourcy CF, De Vlaminck I, Kanbar JN, Wang J, Gawad C and Quake SR: A quantitative comparison of single-cell whole genome amplification methods. PLoS One. 9:e1055852014. View Article : Google Scholar : PubMed/NCBI
20	Nielsen K, Mogensen HS, Hedman J, Niederstätter H, Parson W and Morling N: Comparison of five DNA quantification methods. Forensic Sci Int Genet. 2:226–230. 2008. View Article : Google Scholar : PubMed/NCBI
21	Navarro E, Serrano-Heras G, Castano MJ and Solera J: Real-time PCR detection chemistry. Clin Chim Acta. 439:231–250. 2015. View Article : Google Scholar
22	Singer VL, Jones LJ, Yue ST and Haugland RP: Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation. Anal Biochem. 249:228–238. 1997. View Article : Google Scholar : PubMed/NCBI
23	Robin JD, Ludlow AT, LaRanger R, Wright WE and Shay JW: Comparison of DNA quantification methods for next generation sequencing. Sci Rep. 6:240672016. View Article : Google Scholar : PubMed/NCBI
24	White RA III, Blainey PC, Fan HC and Quake SR: Digital PCR provides sensitive and absolute calibration for high throughput sequencing. BMC Genomics. 10:1162009. View Article : Google Scholar : PubMed/NCBI
25	Panaro NJ, Yuen PK, Sakazume T, Fortina P, Kricka LJ and Wilding P: Evaluation of DNA fragment sizing and quantification by the agilent 2100 bioanalyzer. Clin Chem. 46:1851–1853. 2000.PubMed/NCBI
26	Arya M, Shergill IS, Williamson M, Gommersall L, Arya N and Patel HR: Basic principles of real-time quantitative PCR. Expert Rev Mol Diagn. 5:209–219. 2005. View Article : Google Scholar : PubMed/NCBI
27	Buh Gasparic M, Tengs T, La Paz JL, Holst-Jensen A, Pla M, Esteve T, Zel J and Gruden K: Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection. Anal Bioanal Chem. 396:2023–2029. 2010. View Article : Google Scholar : PubMed/NCBI
28	Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA Jr and Kinzler KW: Cancer genome landscapes. Science. 339:1546–1558. 2013. View Article : Google Scholar : PubMed/NCBI
29	Karro JE, Yan Y, Zheng D, Zhang Z, Carriero N, Cayting P, Harrrison P and Gerstein M: Pseudogene.org: A comprehensive database and comparison platform for pseudogene annotation. Nucleic Acids Res. 35:Database. D55–D60. 2007. View Article : Google Scholar
30	Bhat S, Curach N, Mostyn T, Bains GS, Griffiths KR and Emslie KR: Comparison of methods for accurate quantification of DNA mass concentration with traceability to the international system of units. Anal Chem. 82:7185–7192. 2010. View Article : Google Scholar : PubMed/NCBI
31	Kim IJ, Quigley D, To MD, Pham P, Lin K, Jo B, Jen KY, Raz D, Kim J, Mao JH, et al: Rewiring of human lung cell lineage and mitotic networks in lung adenocarcinomas. Nat Commun. 4:17012013. View Article : Google Scholar : PubMed/NCBI