Abstract
Conventional mitochondrial-DNA (MT DNA) sequencing approaches use Sanger sequencing of 20–40 partially overlapping PCR fragments per individual, which is a time- and resource-consuming process. We have developed a high-throughput, accurate, fast, and cost-effective human MT DNA sequencing approach. In this setup we first generate long-range PCR products for two partially overlapping 7.7 and 9.2 kb MT DNA-specific amplicons, add sample-specific barcodes, and sequence these on the PacBio RSII system to obtain full-length MT DNA sequences for genotyping/haplotyping purposes.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Bartlett JS, Stirling D (2003) A short history of the polymerase chain reaction. Methods Mol Biol 226:3–6
Saiki RK, Gelfand DH, Stoffel S et al (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491
Cheng S, Fockler C, Barnes WM, Higuchi R (1994) Effective amplification of long targets from cloned inserts and human genomic DNA. Proc Natl Acad Sci 91:5695–5699
Shukla SA, Rooney MS, Rajasagi M et al (2015) Comprehensive analysis of cancer-associated somatic mutations in class I HLA genes. Nat Biotechnol 33:1152–1158
Guo X, Lehner K, O’Connell K et al (2015) SMRT sequencing for parallel analysis of multiple targets and accurate SNP phasing. G3 Genes Genomes Genetics 5:2801–2808
Qiao W, Yang Y, Sebra R et al (2016) Long-read single molecule real-time full gene sequencing of cytochrome P450-2D6. Hum Mutat 37:315–323. doi:10.1002/humu.22936
Maitra A, Cohen Y, Gillespie SED et al (2004) The human MitoChip: a high-throughput sequencing microarray for mitochondrial mutation detection. Genome Res 14:812–819
Huang X, Madan A (1999) CAP3: a DNA sequence assembly program. Genome Res 9:868–877
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Vossen, R.H.A.M., Buermans, H.P.J. (2017). Full-Length Mitochondrial-DNA Sequencing on the PacBio RSII. In: White, S., Cantsilieris, S. (eds) Genotyping. Methods in Molecular Biology, vol 1492. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6442-0_12
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6442-0_12
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6440-6
Online ISBN: 978-1-4939-6442-0
eBook Packages: Springer Protocols