Next-Generation Sequencing of Dried Blood Spot Specimens: A Novel Approach to HIV Drug-Resistance Surveillance

Abstract

Background HIV drug-resistance (DR) surveillance in resource-limited settings can be performed using dried blood spots (DBS) because of ease of collection, transportation and storage. Analysis of pooled specimens on next-generation sequencing (NGS)-based platforms, such as the 454 pyrosequencing, is an efficient sequencing method for determining HIV DR rates. In this study, we conducted HIV DR surveillance on DBS using NGS and identified minority variants in individual patients. Methods A total of 48 extracts of DBS from an HIV DR surveillance study in Mexico City were re-amplified using primers tagged with multiplex identifiers, pooled and pyrosequenced. Consensus sequences were generated for each specimen with mixtures identified at positions where >20% of the reads contained a variant. Individual consensus sequences were then analysed for DR mutations and compared with those derived from Sanger sequencing. Results DBS analysed with tagged pooled pyrosequencing (TPP) were highly concordant with Sanger sequencing genotypes from matching plasma and DBS (99.21% and 99.51%, respectively). An exception was an M184I mutation only detected with TPP of DBS at a frequency of 20.4%. Multiple specimens had minority variant reads below the 20% mixture threshold. Conclusions TPP using DBS is an effective method for HIV DR surveillance. TPP for genotyping results in cost savings of 40% over conventional in-house methods. The effect of low-abundance DR mutations, undetectable by conventional methods, remains to be determined. This technology might be applied to any HIV specimen (plasma/ serum) and can also be used for other diagnostic assays where DNA sequencing is required.

Global HIV-1 diversity surveillance

“…The ability to analyse hundreds of patient samples in a single run could make this approach cost-effective if HIV samples are processed and sequenced in a centralised manner. HIVDR surveillance is already being carried out using pyrosequencing of pooled samples, including DBS, from different patients [15]. Tagging of PCR primers can be used to identify individual patient sequences in the output derived from pooled patient samples.…”

Chroniques génomiques

“…Les performances des séquenceurs de nouvelle génération (y compris les « petites » machines comme celle de Ion Torrent/Life Technologies) permettent aussi de revisiter les analyses de pathogènes et d'en proposer des versions beaucoup plus complètes à un tarif accessible. Par exemple, selon une équipe canadienne utilisant le système Roche/454, le séquençage d'ADN viral dans des taches de sang séché permet d'éva-luer les risques de résistance aux antiviraux chez les patients séropo-sitifs [10] ; quant à l'entreprise Pathogenica (Cambridge, États-Unis), elle annonce le lancement d'un test de typage des papillomavirus (HPV) plus sensible et plus fiable que les tests actuels, employant la machine commercialisée par Life Technologies. Bien entendu, comme il s'agit ici d'analyses relativement simples (en termes de quantité de séquence par patient), un multiplexage massif est nécessaire pour que le coût soit concurrentiel.…”

GATA1 Gene Polymorphisms in Down Syndrome Patients

“…The deep sequencing of GATA1 mutations from dried blood spots was carried out in the following manner [17]. Genomic DNA (10 µl) isolated from dried blood spots were applied for GATA1 mutation analysis.…”

“…GATA1 coding regions and their flanking intronic sequences were amplified by polymerase chain reaction (PCR) using the primers (Table 2.) Following PCR, we inserted the barcode for each patient's DNA sample according to user's manual [17]. PCR amplicons were purified and quantified, and the amplicons from patients were pooled at equimolar concentrations with a Beckman Biomex FX system (Beckman, USA) equipped with a DTX880 Multimode Detector (Beckman, USA).…”

“…When using a sensitive method, sequencing the GATA1 mutation by deep sequencing is a good choice. The recent progress in developing high throughput methods for deep sequencing of DNA from dried blood spots and highly sensitive sequencing methods to detect mosaicism makes these the method of choice when detecting GATA1 mutations in DS patients [17,18].…”