Sample Preparation:

CBER/FDA
Primary contact: Viswanath Ragupathy
Sample Preparation:
200ul extraction using Qiagen virus spin kit (Cat:57704)
RNA eluted in 40ul of TE buffer
5ul RNA(~12000cps) used for one step hifi RT PCR (Cat:12574‐030), Two separate PCR’s performed for Pol and Env gene of HIV. No second round PCR performed.
Amplicon size determined by 1% Agarose gel and quantified
by Invitrogen Qubit assay.
1
~2878bp coordinates HXB2 2390‐5267
~1193bp coordinates HXB2 6320‐7512
Two separate one step PCR’s
Amplicons measured with Qubit BR kit (Invitrogen) and normalized to 5ng for illumina sample preparation(Nextera XT).
Pooled and sequenced on Miseq
2
BC Centre for Excellence in HIV/AIDS Laboratory Primary contact: Richard Harrigan Preparation and sequencing of HIV reverse transcriptase and envelope (V3) amplicons from SeraCare panel SeraCare sample preparation SeraCare panel samples 1‐12 were supplied at 5.7 log10 copies/mL. Stock samples were diluted to 5.0 log10 copies/mL and to 4.0 log10 copies/mL HIV‐negative human plasma (SeraCare). To create 90:10 mixtures, samples 1+2, 3+4, 5+6, 7+8, 9+10 and 11+12 were mixed 9:1 starting from the 5.0 log10 copies/mL dilutions. Stock samples and dilutions were stored at ‐20°C until RNA extraction. RNA extraction HIV RNA was extracted from 500 μL of frozen plasma using a NucliSENS easyMAG automated nucleic acid extractor (bioMerieux Canada, St‐Laurent, QC, Canada) per the manufacturer’s instructions. Extracted RNA was eluted in 60 μL elution buffer. If downstream reactions could not be performed immediately, extracted RNA was stored at ‐20°C until RT‐PCR amplification. RT‐PCR amplification of Pol Extracted RNA was reverse transcribed using the Roche Expand Reverse Transcriptase system; In a 10 µL total reaction volume, 4.0 µL of extracted RNA was reverse transcribed using 0.38 µL of 25 µM primer RT3.1 (Table 1). Resulting cDNA was amplified with the Roche High Fidelity PCR system in a set of nested reactions; First round PCR used 10 µL cDNA template, 0.38 µL of 25 µM primer 5CP1 in a 50 µL total reaction volume. A second nested PCR reaction used 2.0 µL template, 0.15 µL each of 25 µM primers 2.5 and RT3798R in a 20 µL total reaction volume. The resulting 1775 bp amplicon spanned the complete protease and RT codons 1‐400. Amplified products were visualized on a 1% agarose gel prior to proceeding with library preparation. RT‐PCR amplification of env (V3) Extracted RNA was reverse transcribed in triplicate using the SuperScript III Reverse Transcriptase ‐ Platinum Taq DNA Polymerase High Fidelity one‐step RT‐PCR kit; 4.0 µL of extracted RNA was amplified using 0.32 µL of each 25 µM primers SQV3F1 and CO602 (Table 2) in a 40 µL total reaction volume. Each replicate was amplified in a second nested PCR reaction using 0.5 µL template, 1 µL each of 2 µM tagged primers ILV3F and ILV3R in a 10 µL total reaction volume. Amplicons were barcoded using a dual‐indexing strategy. Tagged primers incorporated standard Illumina index sequences and adaptors. 3
Nextera XT Library preparation (Pol) Pol amplicons (10 µL) were purified using Agencourt AMPure XP beads (Beckman Coulter Inc). Amplicons concentration was determined by Invitrogen™ Quant‐iT™ PicoGreen® dsDNA Assay (Invitrogen, Inc.). Purified amplicons were diluted to 1.0 ng/µL and processed by Illumina Nextera XT DNA Library Preparation Kit (Illumina Inc.) and uniquely indexed using the Illumina Nextera XT Index Kit (96 Index). Library cleanup followed as described below MiSeq Library Preparation (Env Amplicon; Pol Nextera) A 5 µL aliquot of the library (triplicate env amplicons, or Pol Nextera XT prep) was purified and normalized using a 1:50 dilution of Agencourt AMPure XP beads (Beckman Coulter Inc.) in 15% PEG/2.5M NaCl solution. All recovered eluates (purified env amplicons) were pooled in equal volumes. Pooled amplicons were visualized on a 1.5% agarose gel. DNA concentration of the pool was determined using the Invitrogen™ Quant‐iT™ PicoGreen® dsDNA Assay (Invitrogen, Inc.) MiSeq seqencing Pol and env libraries were pooled at equimolar concentrations and sequenced on the Illumina MiSeq using a 2x250 bp paired‐end kit (version 2). A 10% PhiX spike‐in was used as a control in all sequencing runs. The resulting reads were processed using an in‐house pipeline that incorporated iterative short read mapping with bowtie2 and samtools. Briefly, paired‐end reads were initially mapped to a standard reference (HXB2 for Pol; Consensus B for env). The overlapping portions of paired‐end reads were merged and error correction rules were applied: Bases with sequencing quality scores <15 were discarded; conflicting basecalls in overlapping reads were resolved by retaining the base with the higher quality score. Mapped reads were collapsed into a sample‐specific consensus to which all sequenced reads were iteratively re‐mapped until ≥95% of reads mapped or no additional reads could be recovered by additional rounds of re‐mapping. Samples with <1000‐fold coverage at key resistance positions (pol) or at any position in the V3 loop (env) were excluded from downstream analysis Table 1 – Pol RT‐PCR primers Name Step Direction HXB2 Position Sequence RT3.1 RT Reverse 3858 → 3830 5’ CTCCTACTATGGGTTCTTTCTCTAACTGG 5CP1 PCR1 Forward 1981 → 2008 5’ GAAGGGCACACAGCCAGAAATTGCAGGG 2.5 PCR2 Forward 2011 → 2039 5’ CCTAGGAAAAAGGGCTGTTGGAAATGTGG RT3798R PCR2 Reverse 3798 → 3777 5' CAAACTCCCACTCAGGAATCCA Table 2 – Env (V3) RT‐PCR primers Name Step Direction HXB2 Position Sequence SQV3F1 RT‐PCR1 Forward 6855 → 6878 5’ GAGCCAATTCCCATACATTATTGT CO602 RT‐PCR1 Reverse 7817 → 7786 5’ GCCCATAGTGCTTCCTGCTGCTCCCAAGAACC ILV3F PCR2 Forward 7064 → 7084 5’ AATGCCAAAACCATAATAGTACA ILV3R PCR2 Reverse 7370 → 7350 5' GAAAAATTCCCTTCCACAATTAAA 4
Harvard/BWH
Primary contact: Jon Li
Preparation of SeraCare Proficiency Panel Samples:
All Seracare samples were received at 5x105 copies/mL. Seracare samples 1-12 were extracted
separately at 105 (200ul) and 104 (20ul) HIV copy input. In addition, Samples #1 and #2 were
mixed with sample #1 at 105 (200ul) and sample #2 at 104 (20ul) input copy number. The same
mixing protocol was performed for samples #3 and #4, #5 and #6, and #7 and #8.
Illumina Library Construction and Sequencing
Viral RNA was extracted using the Nuclisens miniMAG protocol. cDNA synthesis was then
performed using Superscript III Reverse Transcriptase for three regions of HIV: ProteaseReverse Transcriptase, Integrase and the V3 loop. We then performed nested PCR using
Platinum Taq Hifi and PCR purified each amplicon. The three amplicons were then pooled for
each sample and processed in the Nextera XT DNA Library Preparation kit. Each sample was
then PCR purified with AMPure XP beads, quantified using qPCR and run on the MiSeq 2x300
paired end runs.
5
RNA Extraction from
plasma
RT-PCR
amplification (PRRT
or ENV)
PCR Cleanup & Normalization
(bead purification)
NGS Sample Prep:
Nextera XT kit
MiSeq
Sequencing
Bfx Analysis &
Reporting
6
•
•
•
•
•
•
•
•
HIV virions were pelleted from plasma and lysed.
RT-PCR reactions were performed on extracted vRNA to
generate HIV PR/RT sequences.
Amplicons were purified and sequencing-ready libraries
were prepared using the Nextera XT DNA Sample
Preparation Kit (Illumina).
Sample libraries underwent 2x150bp paired-end
sequencing on the Illumina MiSeq.
FASTQ files were processed through a custom-designed
automated bioinformatics analysis pipeline.
Briefly, reads are quality trimmed, overlapping paired-end reads
are joined and reads are aligned to a reference in a codon-aware
manner.
Alignments are checked to determine minimal quality metrics:
coverage >10,000X at all positions, >Q30 average phred score at
all positions.
Variants are determined in a codon by codon manner and SNPs
and amino acid variants present at >1% are reported.
7



Illumina MiSeq platform
◦ 2x250bp paired end reads (2x150bp also available)
◦ 24-30 million paired end reads/run
◦ 7.5-8.5+ Gb of data/run
◦ Very high quality sequencing data
 >75% of bases higher than Q30 at 2x250bp
Nextera XT sample prep
◦ 1ng input DNA requirement
 PCR amplicons >300bp
 Plasmids
◦ Barcode and multiplex up to 96 samples/run
Ultra deep sequencing
◦ Coverage >10,000X
◦ Allows detection of variants at or below 1%
8
 Codon‐based data handling
◦ Amino acid changes are explicit, not inferred ex post facto from SNP calls
◦ Codon‐aware re‐alignment for out‐of‐frame codons
 Automated and flexible: pipeline is extensible to any annotated coding region of any virus (HIV, HCV, Flu, RSV, etc.)
 Sensitivity of rare variants down to 0.5%
◦ Pipeline optimized for read depth of ~10,000
◦ Processing time ~10 minutes for 250,000 reads
 “Adaptive alignment” accommodates diverse viruses like HCV
◦ Iterative “alignment, consensus, realignment” strategy to achieve alignment rates of 97 ‐ 99%
 Specialized analyses
◦ E.g.: Identifying APOBEC‐induced hypermutation in proviral DNA samples
 Extensive QC assessment and documentation
◦ NGS read quality, alignment quality, coverage level, strand bias, and insert size (for paired end reads)
9
Quest Diagnostics Primary contact: Ron Kagan  We ran them as‐is, no mixtures, no preparation.  The samples were extracted on the Roche MagnaPURE instrument, reverse‐transcribed and amplified, followed by nested PCR amplification.  Sanger sequencing was performed on an ABI 3730XL and NGS was performed on a Roche/454 GS JR instrument.  Further details of the method have been published in Kagan RM et al. A genotypic test for HIV‐1 tropism combining Sanger sequencing with ultradeep sequencing predicts virologic response in treatment‐experienced patients. PLoS One. 2012;7(9):e46334 (See below for materials and methods section) Study Population and Sample Selection The MOTIVATE 1 and 2 studies were identically designed randomized placebo‐controlled studies to evaluate the safety and efficacy of maraviroc added to optimized background therapy in treatment‐experienced patients. Entry criteria included an R5 tropism result with the original Trofile™ assay [11]. The A4001029 study was a randomized placebo‐controlled phase 2b study to assess the safety and efficacy of maraviroc in treatment‐experienced patients with an X4, dual/mixed or non‐reportable tropism result using the original Trofile assay [13]. MOTIVATE patients screened as dual/mixed, X4 or tropism not determined were offered enrollment in A4001029. A4001029 completed enrollment when the MOTIVATE studies were about 25% accrued. Samples were selected only from patients enrolled in the A4001029 or the MOTIVATE study while the A4001029 study was open to accrual. To be eligible for the present study, patients must have received at least one dose of maraviroc during the A4001029 or the MOTIVATE trial and had tropism screening results of R5, D/M or X4 by the original Trofile assay. Patients whose tropism was undetermined or missing at A4001029 or MOTIVATE study screening were not eligible for this study. There were approximately 125 A4001029 samples and 245 MOTIVATE 1 and 2 samples with sufficient remaining sample volume for tropism reanalysis from patients enrolled in the MOTIVATE study while the A4001029 study was still open. Taken together, this population represents a cohort of patients who received maraviroc as part of an optimized background regimen without regard for coreceptor tropism. Phenotypic Tropism Analysis The Trofile‐ES assay (Monogram Biosciences, San Francisco, CA) was performed retrospectively. R5, X4 or D/M tropism results were obtained for a total of 327 samples. X4 and D/M results were classified together as non‐R5. 10
Triplicate Amplification and Population Sequencing of the V3 Loop Viral RNA was extracted from 0.5 mL of plasma on a MagNA Pure LC automated extraction system using the Large Volume Total Nucleic Acid Isolation Kit (Roche Diagnostics Corp. Indianapolis, IN). Reverse transcription and first‐round PCR was performed with forward and reverse primers SQV3F1 (HXB2 genomic coordinates 6855–6878) and CO602 (HXB2 genomic coordinates 7786–7817) in three independent replicates of 4 uL of extracted viral RNA (total nucleic acid extract), essentially as described in detail elsewhere [48], [49]. A second‐round PCR was then performed using primers customized to contain 3′ adapters necessary for UDS, as detailed in the supplemental materials for Swenson et al [49]. Molecular identifier tags (MIDs) were also incorporated into the primers to allow for amplicons from up to 16 patients to be pooled during UDS. We utilized MID1–13 and MID15–17 (Roche/454 Life Sciences Technical Bulletin 005‐2009, Using Multiplex Identifier (MID) Adaptors for the GS FLX Titanium Chemistry ‐ Extended MID Set). Bidirectional DNA sequencing was performed for all 3 replicates on an ABI 3730XL DNA analyzer (Applied Biosystems, Foster City, CA) using BigDye 3.1 dye terminators with the target‐specific portions of the second round PCR2 primers (forward sequencing primer: HXB2 genomic coordinates 7062–7084; reverse sequencing primer: HXB2 genomic coordinates 7350–7373). Population Sequence Data Analysis and Tropism Interpretation DNA sequence chromatograms were base called and assembled in ReCALL software as described previously [48], [49]. Tropism assignment was performed with the geno2pheno typing program (http://coreceptor.bioinf.mpi‐inf.mpg.de/) [20] and the PSSMx4r5 program (http://fortinbras.us/cgi‐bin/fssm/fssm.pl) [50]. A geno2pheno false‐positive rate (FPR) of ≤5.75% was considered to indicate non‐R5, and sequences with FPR >5.75% were assigned as R5 [29]. For PSSMx4r5 interpretation, sequences scoring ≥−4.75 were assigned as non‐R5 and those scoring <−4.75 were assigned as R5. UDS of V3 loop PCR Amplicons Up to 16 samples and controls with distinct 10‐nt DNA barcodes incorporated into the PCR primers were pooled by combining aliquots of 10 uL of each PCR product. This library was then purified to remove small fragments using Agencourt AMPure XP beads (Agencourt/Beckman Coulter Genomics, Danvers, MA) according to the Roche Amplicon Library Preparation Method Manual. The library was quantitated with the Quant‐iT PicoGreen dsDNA Kit (Invitrogen, Carlsbad, CA) and a SpectraMax Model M2 Spectrofluorometer (Molecular Devices, Sunnyvale, CA). An appropriate dilution of the library was then used to prepare 5 million (GS Junior) or 6.8 million (GS FLX) emulsion PCR (emPCR) A and B microbeads at a ratio of 0.6 to 1.0 molecules of library DNA per microbead. emPCR and bead recovery were then carried out according to theemPCR Amplification Method Manual ‐ Lib‐A GS Junior Titanium Series (Roche/454 Life Sciences, Branford, CT) or the emPCR Amplification Method Manual ‐ Lib‐A MV GS FLX Titanium 11
Series (Roche/454 Life Sciences, Branford, CT). Bead enrichment was performed according to the Roche/454 Life Sciences application note Automated GS FLX Titanium emPCR Enrichment using the REM e System on a Hamilton MICROLAB® STARlet Liquid Handler(http://www.my454.com/my454). UDS was performed on the Roche/454 Life Sciences GS Junior by loading 500,000 beads onto the single‐region pico‐titer plate (PTP) according to the protocol in the Sequencing Method Manual GS Junior Titanium Series (Roche/454 Life Sciences, Branford, CT) or 500,000 beads per region of a four‐region PTP for the GS FLX platform according to the protocol in theSequencing Method Manual GS FLX Titanium Series (Roche/454 Life Sciences, Branford, CT). UDS Sequence Data Analysis and Tropism Assignment The amplicon pipeline was used for both image processing and signal processing (GS Junior system software version 2.5p1 or GS FLX system software version 2.5.3) and then refiltered with optimized amplicon trim filter parameters (doValleyFilterTrimBack = true, vfBadFlowThreshold = 6, vfLastFlowToTest = 480, vfScanAllFlows = false). This process generated median read lengths of 326 nucleotides, including the V3 loop sequence spanning 105 nucleotides, with some minor length variations accounted for by insertions or deletions in the V3 loop. Reads were sorted by MID, dereplicated using the program BARTAB [51] and then split into separate files for the forward and reverse sequencing primer directions. The read replicate count was stored in the fasta header for each sequence to allow for downstream calculation of the proportion of X4 reads. The reads were trimmed to the V3 loop open reading frame (ORF); we defined the ORF as a 90 to 120 nucleotide span within the amplicon starting and ending with a cysteine codon (TG[T/C]), where the length of the span was a multiple of 3 and stop codons (TAG/TAA/TGA) were absent. Reads that did not meet these criteria were excluded from further analysis. The translated amino acid sequences, together with their associated MIDs and replicate counts, were stored in a relational database. The filtered V3 ORFs were processed with the Geno2pheno454 (G2p454) preprocessor and then typed with G2p454 (http://g2p‐
454.bioinf.mpi‐inf.mpg.de/index.php) [52]. The G2p454 scores and alignment scores for each were uploaded to the database. Reads with alignment scores of <75 were excluded, as they likely reflected an improper alignment. V3 ORFs that occurred less than three times were not tabulated and at minimum 400 valid V3 ORFs per sample and 200 reads per forward and reverse direction were required for a tropism assignment. At this coverage, it is theoretically possible to detect a minority variant at the 1% threshold [28]. Reads with an FPR ≤3.5% were classified as X4 and reads with an FPR >3.5% were classified as R5 [29]. Samples found to have ≥2% X4 reads were classified as non‐R5, whereas samples with <2% X4 reads were classified as R5 [29]. 12
The UDS standard flowgram format (SFF) data files for samples in this study have been submitted to the NCBI Short Read Archive (http://www.ncbi.nlm.nih.gov/sra/), submission number SRA056112. Reflex Testing Algorithm All study samples were tested by both population sequencing and UDS. We evaluated a simulated “reflex” approach, whereby the net tropism result was considered to be non‐R5 if any of the TPS replicates had a non‐R5 result. UDS was used to assign tropism only for result only for samples that had an R5 result by TPS. LOD Experiments Mimicked clinical samples consisted of mixtures of the R5 strain US1 (Genbank accession number AY173952) and the X4 strain BK132 (Genbank accession number AY736821). Plasma samples were diluted in Basematrix 53 (Seracare Life Sciences, Milford, MA) to 20%, 15%, 10%, 5% and 2% X4 at a constant viral load of 25,000 copies/mL, and additionally at 5% and 2% X4 at a constant viral load of 100,000 copies/mL. Seven extractions were performed at each level, and triplicate amplifications were carried out to provide 21 replicates per level. Outcome Measures We assessed the ability of each tropism test to predict the median change in log10 plasma viral load (pVL) and virologic response as a function of tropism status at weeks 8 and 24. Virologic response was defined as a viral load of <50 copies/mL or >2 log decline in viral load at week 8, or a viral load of <50 copies/mL at week 24. Immunologic response as defined by changes in CD4(+) T‐cell count at week 24 was also tabulated and categorized by tropism status. Missing virologic outcome data were handled as follows: If a measurement at study entry was missing, the measurement at study screening was used. If a patient discontinued the study prior to week 8 (or week 24), or had a missing value for other reasons, the last available measurement prior to week 8 (or week 24) was carried forward. Patients who did not have any virologic or immunologic measurement after study entry were excluded from the analysis of virologic or immunologic outcome. Statistical Analysis Statistical analysis was performed in SAS (version 9.2, Cary, NC, USA). P‐values for median viral load and CD4(+) changes were estimated using a two‐sided Wilcoxon test. The positive predictive value (PPV) of each assay was defined as the proportion of R5 subjects who achieved a virologic response. Negative predictive value (NPV) was defined as the proportion of non‐R5 subjects who did not achieve a virologic response. 13
Translational Virology Core (UCSD CFAR) Primary contact: Davey Smith Preparation and sequencing of HIV RT and C2‐V3 amplicons from SeraCare panel samples 1‐12 using 454 FLX sequencing platform Preparation of SeraCare samples: SeraCare panel samples 1‐12 were supplied at 5x10e5 copies/ml. Dilutions were not prepared; samples were used at the concentration supplied. Amplicon Generation: Cell‐free virions were concentrated from 0.5ml each of the panel samples by a high speed centrifugation followed by RNA extraction (Roche High Pure Viral RNA kit). In a 20µl reaction volume, 10µl (of 50µl total) HIV‐1 virion RNA was reverse transcribed using Ambion’s RETROscript kit with random decamers. The resulting cDNA (10µl in each PCR) was amplified by nestedPCR for the RT and C2‐V3 env region. Second round primers for the RT were 5RT (5’ AAATCCATACAATACTCCAGTATTTGC) and CI‐185RT_R (5’ CATCCATGTATTGATAGATAACTATGTCTG) resulting in a 397bp amplicon. The second round primers for C2‐V3 were V3‐Fin (5’ GAACAGGACCAGGATCCAATGTCAGCACAGTACAAT) and V3‐Bin (5’ GCGTTAAAGCTTCTGGGTCCCCTCCTGAG) resulting in a 417bp amplicon. Second round PCR products were purified using QIAquick PCR purification kit. Amplicon Sequencing using 454 FLX Amplicons for RT and C2‐V3 were pooled together in equal amounts (125ng of each) for a total of 250ng. Library preps were prepared using GS FLX Titanim Rapid Library Prep Kit (Lib‐L). Emulsions were prepared using GS FLX Titanium SV emPCR Kit (Lib‐L). Sequencing was done on the 454 FLX using the 16‐gasket region. Below is a table of the sample labeling convention: 028551‐1 Prof Panel 1 RT/V3 028551‐2 Prof Panel 2 RT/V3 028551‐3 Prof Panel 3 RT/V3 028551‐4 Prof Panel 4 RT/V3 028551‐5 Prof Panel 5 RT/V3 028551‐6 Prof Panel 6 RT/V3 028551‐7 Prof Panel 7 RT/V3 028551‐8 Prof Panel 8 RT/V3 028551‐9 Prof Panel 9 RT/V3 028551‐10 Prof Panel 10 RT/V3 028551‐11 Prof Panel 11 RT/V3 028551‐12 Prof Panel 12 RT/V3 14
Translational Virology Core (UCSD CFAR) Primary contact: Davey Smith Preparation and sequencing of HIV RT and C2‐V3 amplicons from SeraCare panel samples 1‐12 using the PacBio sequencing platform Preparation of SeraCare samples: SeraCare panel samples 1‐12 were supplied at 5x10e5 copies/ml. Stock samples were diluted to 1x10e5 and 1x10e4 copies/ml using HIV negative plasma. 1.0 mL of each sample 1‐12 at 1x10e5 and 1x10e4 copies/ml was aliquoted and stored at ‐80C until RNA extraction. Amplicon Generation: Cell‐free virions were concentrated from 1ml each of the panel samples by a high speed centrifugation followed by RNA extraction (Roche High Pure Viral RNA kit). In a 20µl reaction volume, 10µl (of 50µl total) HIV‐1 virion RNA was reverse transcribed using Ambion’s RETROscript kit with random decamers. The resulting cDNA (10µl in each PCR) was amplified by nestedPCR for the RT and C2‐V3 env region. Second round primers for the RT were 5RT (5’ AAATCCATACAATACTCCAGTATTTGC) and CI‐185RT_R (5’ CATCCATGTATTGATAGATAACTATGTCTG) resulting in a 397bp amplicon. The second round primers for C2‐V3 were V3‐Fin (5’ GAACAGGACCAGGATCCAATGTCAGCACAGTACAAT) and V3‐Bin (5’ GCGTTAAAGCTTCTGGGTCCCCTCCTGAG) resulting in a 417bp amplicon. Second round PCR products were purified using QIAquick PCR purification kit. Amplicon Sequencing using PacBio Amplicons for RT and C2‐V3 were pooled together in equal amounts (125ng of each) for a total of 250ng. Samples were prepared for sequencing using PacBio SMRT Template Prep Kit 1.0. Library preps were sent to Roche for sequencing on the PacBio platform. Below is a table of the sample labeling convention: 029377‐17 Prof Panel 1 RT/V3 1 x 10x5 Dil 029377‐18 Prof Panel 2 RT/V3 1 x 10x5 Dil 029377‐19 Prof Panel 3 RT/V3 1 x 10x5 Dil 029377‐20 Prof Panel 4 RT/V3 1 x 10x5 Dil 029377‐21 Prof Panel 5 RT/V3 1 x 10x5 Dil 029377‐22 Prof Panel 6 RT/V3 1 x 10x5 Dil 029377‐23 Prof Panel 7 RT/V3 1 x 10x5 Dil 029377‐24 Prof Panel 8 RT/V3 1 x 10x5 Dil 029377‐25 Prof Panel 9 RT/V3 1 x 10x5 Dil 029377‐26 Prof Panel 10 RT/V3 1 x 10x5 Dil 029377‐27 Prof Panel 11 RT/V3 1 x 10x5 Dil 029377‐28 Prof Panel 12 RT/V3 1 x 10x5 Dil 029377‐29 Prof Panel 1 RT/V3 1 x 10x4 Dil 029377‐30 Prof Panel 2 RT/V3 1 x 10x4 Dil 029377‐31 Prof Panel 3 RT/V3 1 x 10x4 Dil 15
029377‐32 029377‐33 029377‐34 029377‐35 029377‐36 029377‐37 029377‐38 029377‐39 029377‐40 Prof Panel 4 RT/V3 Prof Panel 5 RT/V3 Prof Panel 6 RT/V3 Prof Panel 7 RT/V3 Prof Panel 8 RT/V3 Prof Panel 9 RT/V3 Prof Panel 10 RT/V3 Prof Panel 11 RT/V3 Prof Panel 12 RT/V3 1 x 10x4 Dil 1 x 10x4 Dil 1 x 10x4 Dil 1 x 10x4 Dil 1 x 10x4 Dil 1 x 10x4 Dil 1 x 10x4 Dil 1 x 10x4 Dil 1 x 10x4 Dil 16
ARI‐UCSFLaboratoryofClinicalVirology(UCSFCFARVirology)
Primarycontact:TeriLiegler
PreparationandsequencingofHIVreversetranscriptaseampliconsfrom
SeraCarepanel
PreparationofSeraCaresamples:
SeraCarepanelsamples1‐12weresuppliedat5x10e5cps/ml.Stocksampleswere
dilutedto1x10e5and1x10e4cps/mLinBaseMatrixhumanclarifiedplasma
(SeraCare).Tocreate50/50mixtures,samples1+2,3+4,5+6,7+8,9+10and11+12
weremixed1:1.
1.0mLofeachsample1‐12andeach50/50mixturepairateachconcentration
(target1x10e5and1x10e4cps/mL)wasaliquotedandstoredat‐80°CuntilRNA
extraction.
Amplicongeneration(modifiedfromLiegleretal.,JID2014210:1217):
Cell‐freevirionswereconcentratedfrom1mLeachofthepanelsamplesby
centrifugationfollowedbyRNAextraction(QIAampviralRNAkit).Ina40μL
reactionvolume,20.8μL(of60μLtotal)HIV‐1virionRNAwasreversetranscribed
withprimerMM3441.22(5’‐CTGCCAGTTCTAGCTCTGCTTC)at2.5mol/Lusing
theTranscriptorHighFidelitycDNASynthesisKit(RocheAppliedScience).The
resultingcDNAwaspurifiedusingtheHighPurePCRCleanupMicroKit(Roche
AppliedScience)andthetotalproduct(20μL)wasamplifiedbyPCRusingthe
PhusionHigh‐FidelityPCRMasterMixwithHFBuffer(NewEnglandBiolabs)with
primersMM2590.18(5’‐CAGGAATGGATGGCCCAA)andMM3264.24(5’‐CAGCAC
TATAGGCTGTACTGTCCA)resultingina692bpampliconspanningRTdrug
resistancecodonsE44A/DthroughK238S/T.
AmpliconSequencingusingtheIlluminaMiSeq:
Anindexedampliconlibrarywasmadefromtheampliconsgeneratedasdescribe
aboveandsequencedontheGladstoneMiSequsing2x300sequencing.Theresulting
fastqfileswereimportedintoCLCBioGenomicsWorkbenchafterwhichthe300‐
basesequencesweremappedtothesubtypeBconsensusforsubsequentquality‐
basedvariantidentification.Gordonuploadedthefastqfiles,BAMfiles
(representingthealignments),consensussequencesfromthealignments,andVCF
files(representingthevariantreports)tothedatabaseJohannaCraigatGATACA
createdfortheproject.Gordonalsoprovidedherdetailsregardinghowthe
experimentwasdone,soifadditionaldetailsareneeded,Johannawillhaveit.
17
Belowisatableofthesamplelabelingconvention:
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
1A
2A
1A+2A
3A
4A
3A+4A
5A
6A
5A+6A
7A
8A
7A+8A
9A
10A
11A
12A
1B
2B
1B+2B
3B
4B
3B+4B
5B
6B
5B+6B
7B
8B
7B+8B
9B
10B
11B
12B
Stock
BK132
93/US/144
BK132+93/US/144
CM237
US1
CM237+US1
93/US/143
93/US/141
93/US/143+93/US/141
I-2496
CM235
I-2496+CM235
7388
7384
2529
2808
BK132
93/US/144
BK132+93/US/144
CM237
US1
CM237+US1
93/US/143
93/US/141
93/US/143+93/US/141
I-2496
CM235
I-2496+CM235
7388
7384
2529
2808
mix 400 uL stock + 1.6 mL BaseMatrix
mix 400 uL stock + 1.6 mL BaseMatrix
mix 500 uL 1 + 500 uL 2
mix 400 uL stock + 1.6 mL BaseMatrix
mix 400 uL stock + 1.6 mL BaseMatrix
mix 500 uL 1 + 500 uL 2
mix 400 uL stock + 1.6 mL BaseMatrix
mix 400 uL stock + 1.6 mL BaseMatrix
mix 500 uL 1 + 500 uL 2
mix 400 uL stock + 1.6 mL BaseMatrix
mix 400 uL stock + 1.6 mL BaseMatrix
mix 500 uL 1 + 500 uL 2
mix 200 uL stock + 0.8 mL BaseMatrix
mix 200 uL stock + 0.8 mL BaseMatrix
mix 200 uL stock + 0.8 mL BaseMatrix
mix 200 uL stock + 0.8 mL BaseMatrix
mix 100 uL 1 + 900 uL BaseMatrix
mix 100 uL 2 + 900 uL BaseMatrix
mix 100 uL 3 + 900 uL BaseMatrix
mix 100 uL 4 + 900 uL BaseMatrix
mix 100 uL 5 + 900 uL BaseMatrix
mix 100 uL 6 + 900 uL BaseMatrix
mix 100 uL 7 + 900 uL BaseMatrix
mix 100 uL 8 + 900 uL BaseMatrix
mix 100 uL 9 + 900 uL BaseMatrix
mix 100 uL 10 + 900 uL BaseMatrix
mix 100 uL 11 + 900 uL BaseMatrix
mix 100 uL 12 + 900 uL BaseMatrix
mix 100 uL 13 + 900 uL BaseMatrix
mix 100 uL 14 + 900 uL BaseMatrix
mix 100 uL 15 + 900 uL BaseMatrix
mix 100 uL 16 + 900 uL BaseMatrix
Conc
1e5/mL
1e5/mL
1e5/mL
1e5/mL
1e5/mL
1e5/mL
1e5/mL
1e5/mL
1e5/mL
1e5/mL
1e5/mL
1e5/mL
1e5/mL
1e5/mL
1e5/mL
1e5/mL
1e4/mL
1e4/mL
1e4/mL
1e4/mL
1e4/mL
1e4/mL
1e4/mL
1e4/mL
1e4/mL
1e4/mL
1e4/mL
1e4/mL
1e4/mL
1e4/mL
1e4/mL
1e4/mL
Ha
aliquot 1 mL label "1"
aliquot 1 mL label "2"
Label "3"
aliquot 1 mL label "4"
aliquot 1 mL label "5"
Label "6"
aliquot 1 mL label "7"
aliquot 1 mL label "8"
Label "9"
aliquot 1 mL label "10"
aliquot 1 mL label "11"
Label "12"
Label "13"
Label "14"
Label "15"
Label "16"
Label "17"
Label "18"
Label "19"
Label "20"
Label "21"
Label "22"
Label "23"
Label "24"
Label "25"
Label "26"
Label "27"
Label "28"
Label "29"
Label "30"
Label "31"
Label "32"
18
Introduction of Primer ID Approach
Shuntai Zhou
Swanstrom’s lab
UNC‐CH
19
Primer ID Approach to Correct Errors and Recombination
• A stretch of random bases incorporated in the cDNA primer
– 8 bases of “N” makes ~ 65,000 combinations
• Almost all the templates receive a unique Primer ID
• Primer IDs are carried on during all the following amplification and sequencing steps.
• Sequences with same Primer ID are collapsed to create a Primer ID consensus sequence
– Primer ID consensus sequences represent a sample of initial template sequences
20
Primer ID Approach
1 Primer ID = 1 unique viral template at initial sampling
48 = 65536 combination
3’ Reverse compliment
Primer site
NNNNNNNN
5’
5’
Viral RNA
3’
21
MiSeq library construction with Primer ID approach from viral RNA template Primer ID cDNA primer
N8
cDNA synthesis
Viral RNA
Purification
1st Round PCR
N4
Forward Primer with 4 random nucleotides
2nd Round PCR
Purification
Illumina Indexed Primer
Sequencing region
Illumina Library
N8
N4
R1
R2
MiSeq 300 bp pair‐end sequencing
Primer ID Barcode
22
Algorithm of Primer ID Sequencing and Data Analysis
Seracare Samples
RNA extraction
MiSeq library construction
Sequencing Raw sequences
Consensus creation
Primer ID consensus sequences
Align consensus sequences using MUSCLE
Mis‐primed and undetermined sequences
BLAST, Sequence Locator Tool
Filtered Primer ID consensus sequences
23
Other Reference Materials
• Library construction primers
– Env region,
– Protease region,
– RT region
• Library construction protocol
• Consensus creation pipelines (Ruby codes)
• Consensus sequences created by Shuntai Zhou
24
Region
cDNA primer
1st Round PCR forward Primer
HIV‐1 env V1‐V3
GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNCAGTCCATTTTGCTCTACTAATGTTACAATGTGC
GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAGNNNNTTATGGGATCAAAGCCTAAAGCCATGTGT
A
HIV‐1 pol RT
GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAGNNNNGGCCATTGACAGAAGAAAAAATAAAAGC
GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNCAGTCACTATAGGCTGTACTGTCCATTTA
TC
HIV‐1 pol Protease
GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAGNNNNCAGGAGCCGATAGACAAGGAAC
GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNCAGTTTAACTTTTGGGCCATCCATTCC
25
UNC Primary contact: Shuntai Zhou Amplicon Protocol – Version 1.4 09/15/2014 Primer ID cDNA primer
cDNA synthesis
1st Round PCR
Forward Primer with
4 random bases
Illumina Indexed Primer
2nd Round PCR
Sequencing region
Illumina Library
Random bases
Primer ID barcode
Primers (NOTE: HIV‐1 ENV REGION AS AN EXAMPLE) V1F(forwa GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAGNNNNTTATGGGATCAA
rd) AGCCTAAAGCCATGTGTA BV3R Uni GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNCAGTCCATTTT
(Reverse) GCTCTACTAATGTTACAATGTGC Universal Adapter AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAGAGATGTG Indexed CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGC
Adapter TC ADPT_2a GTGACTGGAGTTCAGACGTGTGCTC Note: Primer ID primer and forward primer use random bases. Indexed primers have 24 fixed barcodes. See attached index primer spreadsheet. 26
cDNA synthesis protocol 1. Pipette the following components into a 0.5 ml RNase‐free tube: μl/tube [stock] [final] [mastermix]10x
3.0 dNTP Mix 10 mM each 0.5 1.5 Primer:BV3R Uni 10 μM 0.25 μM 34.5 RNA template 10,000 copies 39.00 Total volume 2. Place tube in 65°C heat block for 3‐5’ (WICDNA program), remove to ice for 1’ (Can put in ice of freezer). 3. Add the following components: μl/tube [stock] [final] [mastermix]12x 12.0 5x buffer 5x 1x 3.0 DTT 100 mM 5 3.0 RNaseOUT 40 u/μl 2 3.0 SSIII RT 200 u/μl 10 21.0 Per tube 4. Mix and incubate at 50°C for 1 hr and then increase to 55°C for 1 hr (WI
program). 5. Inactivate SSIII RT by heating at 70°C for 15’. 6. To each tube, add 1 μl RNase H, incubate at 37°C for 20’. 7. Purify cDNA using Agencourt RNAClean XP a. Resuspend the beads and take an aliquot out. Keep in room temperature for at least 30 minutes before use. (Should be in 1ml aliquots) b. Transfer the cDNA reactions into 1.7 mL RNase‐free tubes. c. Resuspend the beads (Vortex). Add 42 µl of beads to 60 µl cDNA (Ratio: 0.6 – 0.8) Agencourt RNAClean XP beads to each cDNA reaction. d. Mix the Agencourt RNAClean XP and sample thoroughly by pipette mixing 15 times. No votexing. Let the tube incubate at room temperature for 20 minutes before proceeding to the next step. e. Place the tube onto the magnetic tube rack for 5 minutes to separate the beads from solution. f. Slowly aspirate the cleared solution from the tube and discard. This step should be performed while the tube is situated on the rack. Do not 27
disturb the magnetic beads, which have formed a spot on the side of the tube. g. Dispense 500 μL of 70% ethanol into the tube and incubate for 30 seconds at room temperature. Aspirate out the ethanol and discard. Repeat for a total of FOUR washes. It is important to perform these steps with the tube situated on the rack. Do not disturb the separated magnetic beads. Be sure to remove all of the ethanol from the bottom of the well as it may contain residual contaminants. h. Let the reaction tube air‐dry 10 minutes on the rack with the cap open. The tube(s) should air‐dry until the last visible traces of ethanol evaporate. Over drying the sample may result in a lower recovery. i. Remove tube from rack and resuspend beads in 24 µl DNase‐free water by pipetting up and down. (Elution volume can be as low as 15 µl). Place tube back on rack and leave for 3 min. j. Pipette the eluant from the tube while it is situated on the magnetic tube rack. 28
PCR 1 KAPA2G Robust system 1. Complete thaw and votex KAPA reagents (except for enzyme) before use. 2. μl/tube [stock] [final] [mastermix] 10.0 5x Buffer A 5x 1x 10.0 Enhancer 5x 1x 1.0 dNTPs 10 mM 0.2 mM 2.5 V1F 10 µM 0.5 µM 2.5 ADPT_2a 10 µM 0.5 µM 0.5 KAPA Robust polymerase 5 U/µl 2.5 U 23.5 Template cDNA Prepare mastermix in cold box and use repeater pipette to add to each tube in the cold box. Add template cDNA to each tube and pipette up and down to mix. Transfer tubes to a rack in hallway and take upstairs. 3. Cycle (PCR machine #5 SZ ‐> IL): 95˚C 1 min 95˚C 15 s 58˚C 1 min 72˚C 30 s 25 cycles 72˚C 3 min 4˚C On hold 4. Purify PCR products using AmpureXP PCR cleanup kits. a. Vortex the 1 ml aliquot and remove the needed volume. Keep in room temperature for at least 30 minutes before use. b. Transfer the PCR1 reactions into 1.7 mL RNase‐free tubes. c. Resuspend the beads. Add 40 µl (Ratio: 0.6 – 0.8: 1, 36µl – 48µl) Ampure XP beads to each cDNA reaction. d. Mix the Ampure XP and sample thoroughly by votexing. Let the tube incubate at room temperature for 5 minutes before proceeding to the next step (incube off the rack). e. Place the tube onto the magnetic tube rack for 5 minutes to separate the beads from solution. 29
f. Slowly aspirate the cleared solution from the tube and discard. This step should be performed while the tube is situated on the rack. Do not disturb the magnetic beads, which have formed a spot on the side of the tube. g. Dispense 500 μL of 70% ethanol into the tube and incubate for 30 seconds at room temperature. Aspirate out the ethanol and discard. Repeat for a total of two washes. It is important to perform these steps with the tube situated on the rack. Do not disturb the separated magnetic beads. Be sure to remove all of the ethanol from the bottom of the well as it may contain residual contaminants. h. Let the reaction tube air‐dry 10 minutes on the rack with the cap open. The tube(s) should air‐dry until the last visible traces of ethanol evaporate. Over drying the sample may result in a lower recovery. i. Remove tube from rack and resuspend beads in 50 µl DNase‐free water by pipetting up and down. (Elution volume can be as low as 15 µl). Place tube back on rack and leave for 3 min. j. Pipette the 45 µl eluant from the tube while it is situated on the magnetic tube rack. 30
PCR 2 KAPA KAPA HiFi System 1. Complete thaw and votex KAPA reagents (except for enzyme) before use. 2. μl/tube [stock] [final] [mastermix] 5.0 5x KAPA HiFi Fidelity 5x 1x Buffer 1.0 dNTP Mix 10 mM 0.4 mM 1.0 Uni Adapter (ADPT_P1) 10 µM 0.4 µM 0.5 KAPA HiFI polymerase 1 U/µl 0.5 U 1.0 Indexed Adapter 10 µM 0.4 µM 2.0 Template DNA 14.5 Water Prepare mastermix in cold box and use repeater pipette to add to each tube in the cold box. Add Indexed Adapter to each tube. Add template cDNA to each tube and pipette up and down to mix. 3. Cycle (PCR machine #5 SZ ‐> ILM2): 95˚C 2 min 98˚C 20 s 63˚C 15 s 72˚C 30 s 25 ‐ 35 cycles 72˚C 3 min 4˚C On hold 31
Gel Purification and quantification 1. Before gel purification, run 2 µl products on 1% agarose gel to check the bands. 2. Gel purification. (Qiagen QIAquick gel extraction kit) a. Run 2nd round PCR products on 1.2% agarose gel. E = 4 V/cm, T = 60 min. b. Excise DNA fragment. c. Weight the gel; add 3 volume of Buffer QG to 1 volume of gel. d. Incubate at 50 ˚C for 10 minutes to completing dissolve. Vortex every 2‐3 minutes to help dissolve. e. Check the color of gel solution (should be yellow, otherwise add 10 µl 3M sodium acetate). f. Place MinElute column, apply the sample to the column and centrifuge at 1 min. g. Add 500 µl buffer QG and centrifuge for 1 min. h. Repeat step g. once. i.
Add 0.75 ml buffer PE, incubate for 5 minutes at room temperature, centrifuge for 1 minute. j.
Repeat step i. once. k. Discard the fluid, centrifuge for additional 3 minutes. l.
Put the column in a new 1.7 ml tube, add 10 µl buffer EB. Stand for 4 minutes, centrifuge for 2 minutes. 3. Quantification using Invitrogen Qubit dsDNA HS Assay kit. (Q32851). See the attached Qubit dsDNA HS assay protocol. Don’t use Nanodrop to quantify! 4. After quantification, pool libraries in equal amount. Use AMPure XP beads to purify pooled libraries (0.6‐0.8: 1, two washes) if concentration is low. 5. For MiSeq run, it is required that each library has a minimal concentration of 5nM. Higher concentration of library will have more sequencing reads. 32
PCRMixtureofProteaseMutantsfromUNC–ShuntaiZhou
Participatinglabsreceivedtwosetsofcontrols:
·Theshortfragmentsetis400bplongcoveringtheHIV‐1proteaseregions
·Thelongfragmentsetis2890bplongcoveringHIV‐1polgene
·Eachsethas4mixedcontrols,atotalof8tubes.ProficiencyPanelControl
HIV‐1PCRAmpliconMixofProteaseMutants
LongFragments
PP1L,PP2L,PP3L,PP4L
Size:2890bp
Concentration:30.4ng/µLDNAin1xTEbuffer
Primers:
Name
F2215
R5104
HBX2
2215 - 2236
5104 - 5080
5'-3'
CAGGAGCCGATAGACAAGGAAC
TCCATGTGTTAATCCTCATCCTGTC
Referencesequence:(frompNLCH):
>pNLCH_2215to5104
CAGGAGCCGATAGACAAGGAACTGTATCCTTTAGCTTCCCTCAGATCACTCTTTGGCAGCGACCCCTCGT
CACAATAAAGATAGGGGGGCAATTAAAGGAAGCTCTATTAGATACAGGAGCAGATGATACAGTATTAGA
AGAAATGAATTTGCCAGGAAGATGGAAACCAAAAATGATAGGGGGAATTGGAGGTTTTATCAAAGTAAG
ACAGTATGATCAGATACTCATAGAAATCTGCGGACATAAAGCTATAGGTACAGTATTAGTAGGACCTAC
ACCTGTCAACATAATTGGAAGAAATCTGTTGACTCAGATTGGCTGCACTTTAAATTTTCCCATTAGTCCT
ATTGAGACTGTACCAGTAAAATTAAAGCCAGGAATGGATGGCCCAAAAGTTAAACAATGGCCATTGACA
GAAGAAAAAATAAAAGCATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGAAAAATTTCAAAAAT
TGGGCCTGAAAATCCATACAATACTCCAGTATTTGCCATAAAGAAAAAAGACAGTACTAAATGGAGAAA
ATTAGTAGATTTCAGAGAACTTAATAAGAGAACTCAAGATTTCTGGGAAGTTCAATTAGGAATACCACA
TCCTGCAGGGTTAAAACAGAAAAAATCAGTAACAGTACTGGATGTGGGCGATGCATATTTTTCAGTTCC
CTTAGATAAAGACTTCAGGAAGTATACTGCATTTACCATACCTAGTATAAACAATGAGACACCAGGGAT
TAGATATCAGTACAATGTGCTTCCACAGGGATGGAAAGGATCACCAGCAATATTCCAGTGTAGCATGAC
AAAAATCTTAGAGCCTTTTAGAAAACAAAATCCAGACATAGTCATCTATCAATACATGGATGATTTGTA
TGTAGGATCTGACTTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAACTGAGACAACATCTGTTGAG
GTGGGGATTTACCACACCAGACAAAAAACATCAGAAAGAACCTCCATTCCTTTGGATGGGTTATGAACTC
CATCCTGATAAATGGACAGTACAGCCTATAGTGCTGCCAGAAAAGGACAGCTGGACTGTCAATGACATA
CAGAAATTAGTGGGAAAATTGAATTGGGCAAGTCAGATTTATGCAGGGATTAAAGTAAGGCAATTATGT
AAACTTCTTAGGGGAACCAAAGCACTAACAGAAGTAGTACCACTAACAGAAGAAGCAGAGCTAGAACTG
33
GCAGAAAACAGGGAGATTCTAAAAGAACCGGTACATGGAGTGTATTATGACCCATCAAAAGACTTAATA
GCAGAAATACAGAAGCAGGGGCAAGGCCAATGGACATATCAAATTTATCAAGAGCCATTTAAAAATCTG
AAAACAGGAAAGTATGCAAGAATGAAGGGTGCCCACACTAATGATGTGAAACAATTAACAGAGGCAGTA
CAAAAAATAGCCACAGAAAGCATAGTAATATGGGGAAAGACTCCTAAATTTAAATTACCCATACAAAAG
GAAACATGGGAAGCATGGTGGACAGAGTATTGGCAAGCCACCTGGATTCCTGAGTGGGAGTTTGTCAAT
ACCCCTCCCTTAGTGAAGTTATGGTACCAGTTAGAGAAAGAACCCATAATAGGAGCAGAAACTTTCTAT
GTAGATGGGGCAGCCAATAGGGAAACTAAATTAGGAAAAGCAGGATATGTAACTGACAGAGGAAGACAA
AAAGTTGTCCCCCTAACGGACACAACAAATCAGAAGACTGAGTTACAAGCAATTCATCTAGCTTTGCAGG
ATTCGGGATTAGAAGTAAACATAGTGACAGACTCACAATATGCATTGGGAATCATTCAAGCACAACCAG
ATAAGAGTGAATCAGAGTTAGTCAGTCAAATAATAGAGCAGTTAATAAAAAAGGAAAAAGTCTACCTGG
CATGGGTACCAGCACACAAAGGAATTGGAGGAAATGAACAAGTAGATAAATTGGTCAGTGCTGGAATCA
GGAAAGTACTATTTTTAGATGGAATAGATAAGGCCCAAGAAGAACATGAGAAATATCACAGTAATTGGA
GAGCAATGGCTAGTGATTTTAACCTACCACCTGTAGTAGCAAAAGAAATAGTAGCCAGCTGTGATAAAT
GTCAGCTAAAAGGGGAAGCCATGCATGGACAAGTAGACTGTAGCCCAGGAATATGGCAGCTAGATTGTA
CACATTTAGAAGGAAAAGTTATCTTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTAA
TTCCAGCAGAGACAGGGCAAGAAACAGCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAA
CAGTACATACAGACAATGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTGGGCGGGGAT
CAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAATCTATGAATAAAGAATT
AAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATT
CATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAAT
AGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTA
CAGGGACAGCAGAGATCCAGTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAGGTGAAGGGGCAGTAGT
AATACAAGATAATAGTGACATAAAAGTAGTGCCAAGAAGAAAAGCAAAGATCATCAGGGATTATGGAA
AACAGATGGCAGGTGATGATTGTGTGGCAAGTAGACAGGATGAGGATTAACACATGGA
34
ProficiencyPanelControl
HIV‐1PCRAmpliconMixofProteaseMutants
ShortFragments
PP1S,PP2S,PP3S,PP4S
Size:400bp
Concentration:21.0ng/µLDNAin1xTEbuffer
Primers:
Name
F2215
R2614
HBX2
2215 - 2236
2614 - 2592
5'-3'
CAGGAGCCGATAGACAAGGAAC
TTAACTTTTGGGCCATCCATTCC
Referencesequence:(FrompNLCH)
>pNLCH_2215to2614
CAGGAGCCGATAGACAAGGAACTGTATCCTTTAGCTTCCCTCAGATCACTCTTTGGCAGCGACCCCTCGT
CACAATAAAGATAGGGGGGCAATTAAAGGAAGCTCTATTAGATACAGGAGCAGATGATACAGTATTAGA
AGAAATGAATTTGCCAGGAAGATGGAAACCAAAAATGATAGGGGGAATTGGAGGTTTTATCAAAGTAAG
ACAGTATGATCAGATACTCATAGAAATCTGCGGACATAAAGCTATAGGTACAGTATTAGTAGGACCTAC
ACCTGTCAACATAATTGGAAGAAATCTGTTGACTCAGATTGGCTGCACTTTAAATTTTCCCATTAGTCCT
ATTGAGACTGTACCAGTAAAATTAAAGCCAGGAATGGATGGCCCAAAAGTTAA
35