CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

592 Within-Run Cross-Contamination in Deep Sequencing Applications on the Illumina MiSeq Chanson J. Brumme ;Winnie Dong; Celia K. Chui; Richard Liang; Art F. Poon; Richard Harrigan BC Centre for Excellence in HIV/AIDS, Vancouver, Canada

Background: The Illumina MiSeq DNA sequencing system generates several gigabases of short reads per run with a relatively low error rate. We previously described longitudinal contamination on this platformwhich has since been addressed by a post-run bleach wash. Here we characterize rates and sources of systematic low level, within-run cross-sample contamination, an under-reported issue for this platform. Methods: In order to assess cross-contamination observed in previous experiments, two libraries of disparate amplicons (HCV NS5B, human HLA-B) were sequenced at high read depth on a single MiSeq run (v2, 2x250bp). HCV RNA was extracted from 24 patient-derived plasma samples using a NucliSens easyMag, and a 327-bp fragment of NS5B was amplified by nested RT-PCR. Human genomic DNA was extracted from 33 whole blood samples and a region spanning HLA-B exons 2 and 3 was amplified. All stages of HCV and HLA library preparation were performed on different days by different staff. Indexed PCR primers for these targets were ordered months apart, effectively ruling out primer synthesis as a source of cross-contamination. Including replicates, 69 amplicons were sequenced using a total of 56 Illumina index pairs. Sequenced HCV and HLA samples shared either zero, one or two indices with samples of the opposite type. Short read data were cleaned and iteratively mapped using a custom pipeline built around bowtie2 and samtools . Results: The run cluster density was 940 K/mm 2 with 89% of reads passing filters, suggesting normal instrument performance and library preparation. On average, approximately 141,000, and 177,000-fold coverage was obtained for HCV and HLA-B, respectively. Interestingly, up to 3637 HLA-B reads (1.8% of total reads) were observed in samples expected to contain only HCV, and up to 217 HCV reads (0.09%) were observed in HLA-B samples. Screening all suspected contaminants (e.g. HCV reads in an HLA sample) against all consensus sequences indicated that the source of contamination was far more likely to be a sample that shared one Illumina index than a sample that shared none (OR=15.7, p=10 - 11 ). Cross-contamination between reads sharing one index was also observed between samples of the same type. Conclusions: The MiSeq is subject to low-level cross-contamination from samples that share one “barcode” in a dual-indexing strategy. Accurate interpretation of low-frequency variants detected by deep sequencing requires knowledge of all other samples run on the instrument and their associated barcodes. 593 Analysis of Resistance Haplotypes Using Primer IDs and Next Gen Sequencing of HIV RNA Valerie F. Boltz 1 ; Jason Rausch 1 ;Wei Shao 2 ; Charles Coomer 1 ; JohnW. Mellors 3 ; Mary Kearney 1 ; John M. Coffin 4 1 National Institutes of Health (NIH), Frederick, MD, US; 2 Leidos, Frederick, MD, US; 3 University of Pittsburgh, Pittsburgh, PA, US; 4 Tufts University, Boston, MA, US Background: Targeted sequencing technologies using primer IDs can result in more accurate representations of HIV-1 populations but PCR bias and recombination have hampered progress. Here we describe a newmethod for library construction that produces a larger number of tagged consensus sequences, increases sensitivity of haplotype determination, and reveals the sources of recombination. Methods: Each molecule of cDNA frommixtures of varying percentages of wild-type and mutant HIV-1 pol transcripts containing 14 drug resistance mutations was tagged uniquely using a gene-specific primer with primer IDs. cDNAs were then PCR amplified using two methods: (1) 90mer primers containing required MiSeq sequences; (2) 22mer primers containing uracil followed by digestion, cleavage and ligation to linkers containing MiSeq sequences. DNA was sequenced using paired-end MiSeq Illumina technology and consensus sequences were derived from a super-majority ( ≥ 80% consensus) for each unique ID. Consensus sequences were analyzed for PCR bias, errors, recombination, and sensitivity for detecting haplotypes. Results: Of the total cDNA molecules used as template, amplified cDNA with unique tags ranged from 3-19% for method 1 and from 15-52% for method 2. The average error rates for method 1 and 2 were 9.3x10 -5 and 1.4x10 -4 , respectively, both comparable to RT error rates. The PCR recombination rate for method 1 was 0.16% but only 0.01% for method 2. Method 1 was able to detect drug resistance mutations down to 0.01% and method 2 down to 0.001%. The sensitivity of haplotype detection was better for method 2: for samples containing 10% or 1%mutant, method 1 never detected linkage of all 14 mutations, whereas method 2 detected all 14 33-35% of the time. Method 2 always detected linkage of the 8 mutations nearest the 3’ end of the amplicon suggesting that PCR recombination is due to incomplete cDNA synthesis. Conclusions: A linker ligation method of amplifying tagged cDNA reduced both PCR bias and recombination rate compared to standard methods, and was superior at detecting haplotypes within 200bp of the 3’ end of the template. However, it correctly detected linkage across the entire 570bp amplicon in only 1/3 of sequences, suggesting that cDNA synthesis is typically incomplete leading to PCR recombination and thus limiting sensitivity for detection of linked mutations. Improved methods are needed for cDNA synthesis to increase the reliability of haplotype determination for HIV-1 populations. 2:30 pm– 4:00 pm HIV Subtypes and Resistance 594 HIV-1 Subtype Influences the Pathways of Genotypic Resistance to Integrase Inhibitors Tomas J. Doyle 1 ; David Dunn 4 ; Rolf Kaiser 3 ; Erasmus Smit 10 ; Anne-Genevieve Marcelin 5 ; Carmen de Mendoza 6 ; Javier Martínez-Picado 9 ; Federico Garcia 7 ; Francesca Ceccherini-Silberstein 8 ; Anna Maria Geretti 2 CORONET study group 1 King’s College London, London, United Kingdom; 2 University of Liverpool, Liverpool, United Kingdom; 3 University of Cologne, Cologne, Germany; 4 University College London, London, United Kingdom; 5 Hôpital Pitié Salpetriere, APHM, Paris, France; 6 Hospital Carlos III, Madrid, Spain; 7 University Hospital San Cecilio, Granada, Spain; 8 University of Rome Tor Vergata, Rome, Italy; 9 Institut de Recerca de la Sida, Barcelona, Spain; 10 Birmingham Heartlands Hospital, Birmingham, United Kingdom Background: The mutational profiles selected in vivo by integrase inhibitors (INIs) have been primarily defined within clinical trials that involved predominantly subjects with HIV-1 subtype B infection. CORONET is multicentre surveillance programme that aims to characterise INI-resistance in diverse INI-naïve and INI-experienced European cohorts. This analysis addressed the influence of HIV-1 subtype on pathways of genotypic resistance and cross-resistance observed in raltegravir-experienced patients. Methods: Integrase sequences produced by Sanger sequencing at 9 clinical centres were analysed centrally to identify major INI resistance-associated mutations (RAMs) as defined by the Stanford interpretation algorithm. Codon usage at major integrase resistance codons was analysed using integrase sequences from INI-naïve patients. Proportions were compared by Fisher’s Exact test (2-sided). Results: Sequences were examined from 255 raltegravir-experienced (subtype B 209/255, 82%) and 533 raltegravir-naïve patients (subtype B 399/533, 75%). Non-B subtypes comprised 11 different variants: the most prevalent were subtype C and CRF02; other variants included A, D, F, G, CRF01, CRF06 and CRF09. Overall 113 (44%) raltegravir- THURSDAY, FEBRUARY 26, 2015 Session P-L2 Poster Session Poster Hall

Poster Abstracts

377

CROI 2015