Challenges with using primer IDs to improve accuracy of next generation sequencing

PloS One
Johanna BrodinJan Albert

Abstract

Next generation sequencing technologies, like ultra-deep pyrosequencing (UDPS), allows detailed investigation of complex populations, like RNA viruses, but its utility is limited by errors introduced during sample preparation and sequencing. By tagging each individual cDNA molecule with barcodes, referred to as Primer IDs, before PCR and sequencing these errors could theoretically be removed. Here we evaluated the Primer ID methodology on 257,846 UDPS reads generated from a HIV-1 SG3Δenv plasmid clone and plasma samples from three HIV-infected patients. The Primer ID consisted of 11 randomized nucleotides, 4,194,304 combinations, in the primer for cDNA synthesis that introduced a unique sequence tag into each cDNA molecule. Consensus template sequences were constructed for reads with Primer IDs that were observed three or more times. Despite high numbers of input template molecules, the number of consensus template sequences was low. With 10,000 input molecules for the clone as few as 97 consensus template sequences were obtained due to highly skewed frequency of resampling. Furthermore, the number of sequenced templates was overestimated due to PCR errors in the Primer IDs. Finally, some consensus template sequences were erron...Continue Reading

References

Mar 23, 2004·Nucleic Acids Research·Robert C Edgar
Jun 20, 2007·Nucleic Acids Research·Christian HoffmannFrederic D Bushman
Jul 31, 2007·Genome Biology·Susan M HuseDavid Mark Welch
Feb 13, 2009·The Journal of Infectious Diseases·Birgitte B SimenUNKNOWN Terry Beirn Community Programs for Clinical Research on AIDS
Jan 19, 2010·Nature Methods·Joseph B HiattJay Shendure
Jul 31, 2010·Nucleic Acids Research·Osvaldo ZagordiNiko Beerenwinkel
Feb 1, 2011·BMC Bioinformatics·Christopher QuincePeter J Turnbaugh
Jun 21, 2011·Bioinformatics·Susanne BalzerInge Jonassen
Nov 22, 2011·Nature Methods·Teemu KiviojaJussi Taipale
Dec 3, 2011·Proceedings of the National Academy of Sciences of the United States of America·Cassandra B JabaraRonald Swanstrom
Jan 11, 2012·Proceedings of the National Academy of Sciences of the United States of America·Katsuyuki ShiroguchiX Sunney Xie
Jul 6, 2013·Science·Jeremiah J FaithJeffrey I Gordon

❮ Previous
Next ❯

Citations

Dec 30, 2015·Journal of Molecular Biology·David SeifertKarin J Metzner
Aug 5, 2016·Nature Protocols·M A TurchaninovaD M Chudakov
Feb 24, 2018·Molecular Biology and Evolution·William S DeWittFrederick A Matsen
Mar 8, 2018·Nucleic Acids Research·Morgane BooneNico Callewaert

❮ Previous
Next ❯

Methods Mentioned

BETA
454
PCR
454 sequencing
PCRs
454-sequencing

Software Mentioned

house
Primer ID
Sequence Locator Tool
Python scripts
Primer ID UDPS
in
Ion
BioEdit
Primer
Muscle

Related Concepts

Related Feeds

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.