Papers of Note from In Sequence, Mar 2009 (11)

Quantification of rare allelic variants from pooled genomic DNA.
Todd E Druley, Francesco L M Vallania, Daniel J Wegner, Katherine E Varley, Olivia L Knowles, Jacqueline A Bonds, Sarah W Robison, Scott W Doniger, Aaron Hamvas, F Sessions Cole, Justin C Fay, Robi D Mitra.
Nature Methods 6, 263-265 (2009) | doi:10.1038/nmeth.1307 | PMID:19252504

We report a targeted, cost-effective method to quantify rare single-nucleotide polymorphisms from pooled human genomic DNA using second-generation sequencing. We pooled DNA from 1,111 individuals and targeted four genes to identify rare germline variants. Our base-calling algorithm, SNPSeeker, derived from large deviation theory, detected single-nucleotide polymorphisms present at frequencies below the raw error rate of the sequencing platform.

---

Low‐Abundance Drug‐Resistant Viral Variants in Chronically HIV‐Infected, Antiretroviral Treatment–Naive Patients Significantly Impact Treatment Outcomes.
Birgitte B. Simen, Jan Fredrik Simons, Katherine Huppler Hullsiek, Richard M. Novak, Rodger D. MacArthur, John D. Baxter, Chunli Huang, Christine Lubeski, Gregory S. Turenchalk, Michael S. Braverman, Brian Desany, Jonathan M. Rothberg, Michael Egholm, Michael J. Kozal.
The Journal of Infectious Diseases 199, 693–701 (2009) | DOI: 10.1086/596736 | PMID:19210162

Background. Minor (i.e., <20% prevalence) drug‐resistant human immunodeficiency virus (HIV) variants may go undetected, yet be clinically important. Objectives. To compare the prevalence of drug‐resistant variants detected with standard and ultra‐deep sequencing (detection down to 1% prevalence) and to determine the impact of minor resistant variants on virologic failure (VF).

Methods. The Flexible Initial Retrovirus Suppressive Therapies (FIRST) Study (N = 1397) compared 3 initial antiretroviral therapy (ART) strategies. A random subset (n = 491) had baseline testing for drug‐resistance mutations performed by use of standard sequencing methods. Ultra‐deep sequencing was performed on samples that had sufficient viral content (N = 264). Proportional hazards models were used to compare rates of VF for those who did and did not have mutations identified.

Results. Mutations were detected by standard and ultra‐deep sequencing (in 14% and 28% of participants, respectively; P<0.001 ). Among individuals who initiated treatment with an ART regimen that combined nucleoside and nonnucleoside reverse‐transcriptase inhibitors (hereafter, "NNRTI strategy"), all individuals who had an NNRTI‐resistance mutation identified by ultra‐deep sequencing experienced VF. When these individuals were compared with individuals who initiated treatment with the NNRTI strategy but who had no NNRTI‐resistance mutations, the risk of VF was higher for those who had an NNRTI‐resistance mutation detected by both methods (hazard ratio [HR], 12.40 [95% confidence interval {CI}, 3.41–45.10]) and those who had mutation(s) detected only with ultra‐deep sequencing (HR, 2.50 [95% CI, 1.17–5.36]). Conclusions. Ultra‐deep sequencing identified a significantly larger proportion of HIV‐infected, treatment‐naive persons as harboring drug‐resistant viral variants. Among participants who initiated treatment with the NNRTI strategy, the risk of VF was significantly greater for participants who had low‐ and high‐prevalence NNRTI‐resistant variants.

---

Simultaneous mutation and copy number variation (CNV) detection by multiplex PCR-based GS-FLX sequencing.
Dirk Goossens, Lotte N. Moens, Eva Nelis, An-Sofie Lenaerts, Wim Glassee, Andreas Kalbe, Bruno Frey, Guido Kopal, Peter De Jonghe, Peter De Rijk, Jurgen Del-Favero.
Human Mutation 30, 472-476 (2009) | doi:10.1002/humu.20873 | PMID:19058222

We evaluated multiplex PCR amplification as a front-end for high-throughput sequencing, to widen the applicability of massive parallel sequencers for the detailed analysis of complex genomes. Using multiplex PCR reactions, we sequenced the complete coding regions of seven genes implicated in peripheral neuropathies in 40 individuals on a GS-FLX genome sequencer (Roche). The resulting dataset showed highly specific and uniform amplification. Comparison of the GS-FLX sequencing data with the dataset generated by Sanger sequencing confirmed the detection of all variants present and proved the sensitivity of the method for mutation detection. In addition, we showed that we could exploit the multiplexed PCR amplicons to determine individual copy number variation (CNV), increasing the spectrum of detected variations to both genetic and genomic variants. We conclude that our straightforward procedure substantially expands the applicability of the massive parallel sequencers for sequencing projects of a moderate number of amplicons (50-500) with typical applications in resequencing exons in positional or functional candidate regions and molecular genetic diagnostics.