Clc genomics workbench version 5

Library construction and paired-end sequencing were performed at the Centro Nacional de Análisis Genómico (CNAG-CRG, Spain; http://www.cnag.crg.eu/ accessed on 5 November 2015) using Illumina HiSeq 2000 technology. A total of 52,845,525 paired-end, 2 × 126 nucleotides (nt) sequence reads were generated. A median insert size of 305-bp was estimated. The reads were analyzed using PrinseqQuality (http://prinseq.sourceforge.net/ accessed on 3 March 2016) and poor-quality reads (cut-off value, 20) were removed; additionally, only those reads having a length ≥60-nt were considered. The filtered reads were assembled using the CLC Genomics Workbench version 5.0 (CLC Bio).

Genome Analyzer IIx of the Illumina-Solexa platform at the Biomedical Genomics Research Center of Korea Research Institute of Bioscience and Biotechnology was used for genome sequencing. 22,525,438 high-quality reads with 233-fold coverage for NCCP15655 and 27,858,714 high-quality reads with 235-fold coverage for NCCP15656 were generated from 500-bp paired-end libraries. Sequence trimming and de novo assembly were performed using CLC Genomics Workbench version 5.1 (CLC bio, Inc.) and scaffolding was carried out with SSPACE [17 (link)]. Automatic gap filling was performed using IMAGE [18 (link)] and manual gap filling was performed using CLC Genomics Workbench. Structural gene prediction was performed using Glimmer 3 [19 (link)] and functional annotation was performed using blastp against MicroScope database [20 (link)] of E. coli and Shigella species. We then employed automatic annotation using the RAST server [21 (link)] and compared it with the annotation result from MicroScope database for more accurate functional assignment. We also performed additional blastp against the subsystem database of the RAST server for the gene categorization.

UFV-P2 genome was sequenced using an Illumina Genome Analyzer II by CD Genomics (New York, USA) and was assembled and analyzed using CLC Genomics Workbench version 5.1 (CLC bio, Cambridge, MA, USA). The sequence reads were assembled into contigs using stringent parameters, in which 90% of each read had to cover the other read with 90% identity. The data are available in GenBank database under accession number JX863101.

For provirus sequences generated in this study, the MPS reads of partial pol gene associated with DRMs in the protease and reverse transcriptase regions of the HIV-1 genome of each sample were aligned to their corresponding consensus sequence using the CLC Genomics Workbench version 5.5 (CLC Bio, Aarhus, Denmark). The minority HIV-1 resistant variants were identified using a threshold of >1.0% of the reads sequenced. Reads with <1% were discarded to account for potential errors due to the error rate of PCR. Amino acid positions including all listed major mutations and minor mutations associated with drug resistance were identified according to the IAS-USA 2011 and Stanford HIV drug resistance database. Due to the polymorphic nature of most minor protease substitutions, we only considered major mutations as evidence of transmission of drug resistance. The list of the NRTIs resistance related sites 41, 62, 65, 67, 69, 70, 71, 74, 75, 77, 115, 116, 151, 184, 210, 215 and 219; NNRTIs resistance related sites 90, 98, 100, 101, 103, 106, 108, 138, 179, 181, 188, 190,221, 225, 227, 230; PIs resistance related sites 10, 16, 11, 20, 24, 30, 32, 33, 35, 36, 43, 46, 47, 48, 50, 53, 54, 58, 60, 62, 63, 64, 69, 71, 73, 74, 76, 77, 82, 83, 84, 85, 88, 89, 90 and 93. Bolded numbers are major drug resistance mutation sites.

Fastq files were generated by the Illumina MiSeq reporter for downstream analysis and validated to evaluate the distribution of quality scores and to ensure that quality scores do not drastically drop over each read. To take the sequencing error rate into account, we only considered variants detected at a frequency higher than 1% and Phred quality score of >30, i.e., a base call accuracy of 99.9%. Validated fastq files from each viral genome were de novo assembled into contiguous sequences and annotated with CLC Genomics Workbench version 5.5 (CLC Bio, Aarhus, Denmark) with default parameters. The contiguous genomic sequence from each virus strain was extracted from the assembly and used for further analysis. Phylogenetic relationships of the newly generated consensus sequences were determined from sequence comparisons with previously published representatives of HIV-1 group M. The full designation of samples, according to WHO-proposed nomenclature, is YYBRCY_XXX, where YY stands for the year of study, BR for Brazil, CY stands for city of enrolment; XXX stands for sample number.

One ml blood plasma from each animal was filtered (0.45 µm) to remove residual host cells, and viral RNA was isolated using the Qiagen QIAamp MinElute virus spin kit (Qiagen, Hilden, Germany), omitting carrier RNA. The eluted RNA was treated with DNase I (DNA-free, Ambion, Austin, TX, USA), and double-stranded DNA was generated using the Superscript double-stranded cDNA Synthesis kit (Invitrogen, Carlsbad, CA, USA), primed with random hexamers. The DNA was purified using the Agencourt Ampure XP system (Beckman Coulter, Brea, CA, USA) and approximately 1 ng DNA was prepared for sequencing on an Illumina MiSeq (Illumina, San Diego, CA, USA) using the Nextera DNA sample preparation kit (Illumina, San Diego, CA, USA). Sequence data were analyzed using CLC Genomics Workbench version 5.5 (CLC bio, Aarhus, Denmark). Briefly, low-quality (CLC quality trimming limit = 0.001; phred quality score <30) and short reads (<100 bp) were removed and the remaining reads were subjected to de novo assembly. Assembled contiguous sequences (contigs) were queried against the GenBank database using the basic local alignment search tools blastn and blastx.