Challenges with using primer IDs to improve accuracy of next generation sequencing.
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 erroneous due to hotspots for UDPS errors. The Primer ID methodology has the potential to provide highly accurate deep sequencing. However, it is important to be aware that there are remaining challenges with the methodology. In particular it is important to find ways to obtain a more even frequency of resampling of template molecules as well as to identify and remove artefactual consensus template sequences that have been generated by PCR errors in the Primer IDs.
Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
Johanna
J
Brodin
Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
Charlotte
C
Hedskog
Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
Alexander
A
Heddini
Max Planck Institute for Developmental Biology, Tuebingen, Germany.
Emmanuel
E
Benard
Max Planck Institute for Developmental Biology, Tuebingen, Germany.
Richard A
RA
Neher
Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden; Unit for Support, Swedish Institute for Communicable Disease Control, Stockholm, Sweden.
Mattias
M
Mild
Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden; Department of Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden.
Jan
J
Albert
D001483:Base Sequence; D017931:DNA Primers; D015497:HIV-1 / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017421:Sequence Analysis / Q000379:methods* / Q000592:standards; D012689:Sequence Homology, Nucleic Acid
D017931:DNA Primers