TITLE: Nextera XT protocol for MiSeq HIVPR-RT sequencing
AUTHORS: Paula  Aulicino

[DESCRIPTION]
The protocol allows amplification and NGS sequencing of a PR-RT HIV-1 sequence using tagmentation strategy and Illumina MiSeq equipment, followed by analysis of drug-resistance associated mutations by HyDRA

[STEPS]
1. Prepare the sample in BSL2 laboratory. This protocol can be used to amplify and sequence HIV-1 from plasma or culture supernatants, as well as from cell-associated HIV-1. In the latter, skip the step of reverse transcription and go straight to PCR amplification.
2. Perform reverse transcription of HIV-1 and PCR amplification of PR-RT 1168bp fragment using primers and conditions published in Aulicino et al, Antivir Ther 2010;15(4):641-50. PCR amplifications are performed in 25-cycle reactions to avoid introducing artificial mutations.
 

3. Check second PCR products with agarose gel electrophoresis (1%). A single 1168bp band should be visible.
4. Perform purification of the PCR product with magnetic beads (Agencourt AMPure XP kit, Beckman Coulter, USA)
5. Perform tagmentation and indexation with Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA), following manufacturer´s instructions.
6. Check electrophoresis tagmentation patterns with QIAxel or another similar equipment. If none is available, a polyacrylamide gel can be used.
7. Quantify DNA using the fluorometric Qubit dsDNA high-sensitivity (HS) assay (Qubit 1X dsDNA HS Assay Kit, ThermoFisher Scientific, USA)
8. Calculate PCR samples concentration and volume to normalize all samples to 2nM, using PCR water.
9. Pool all samples using 5ul of each 2nM dilution of each sample.
10. Prepare PhiX at 12.5 pM. Dilute the library to 8 picomolar in HT1 buffer, and add 20% of PhiX. The purpose of PhiX is to add diversity to the samples.
11. Run in Illumina MiSeq
 

SECTION: Controls and Error Rate calculation
12. pNL4-3 HIV-1 was used as control. Using BWA, align fastq reads belonging to the control sample to the reference pNL4-3 HIV-1 genome (GenBank Accession Number AF324493). Calculate the average number of mismatches/ total bases. Error Rate should not exceed 0,5%. 
SECTION: Sequence analysis using HyDRA
13. HyDRA (https://hydra.canada.ca/pages/home?lang=en-CA) is an annotated reference-based bioinformatics pipeline scripted in Perl, which analyses next generation sequencing (NGS) data for genotyping HIV-1 drug resistance (HIVDR) mutations. It utilizes an annotated HXB2 sequence (GenBank Accession number: K03455) for reference mapping by Bowtie2 2 , and stringent data quality assurance and variant calling criteria to identify HIVDR associated mutations based on the Stanford HIV Drug Resistance Database (http://hivdb.stanford.edu/) and 2009 WHO list for Surveillance of Transmitted HIVDR. All HIVDR mutations found in the pol genes; protease (PR), reverse transcriptase (RT), and integrase (IN), are reported according to classifications outlined in the Stanford Surveillance Drug Resistance Mutation list .
 

SECTION: Sequence analysis using HyDRA
14. Submit sequences to HyDRA. The following Advanced options for Nextera seqs can be used to inform amino acids in 1% frequency or higher, with a minimum read depth of 1000 x:
Consensus percent: 20
Target coverage: -1
Length cutoff: 100
Score cutoff: 2
Error rate: include error rate calculated with the control (e.g. 0.005)
Minimum variant quality: 20
Minimum read depth: 1000
Minimum allele count: 7
Minimum amino acid frequency: 0.01