viroseq hiv-1 genotype testing with the new …€¦ · viroseq™ hiv-1 genotype testing with the...
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VIROSEQ™ HIV-1 GENOTYPE TESTING WITH THE NEW ABI PRISM® 3100 GENETIC ANALYZERP BAYBAYAN, B HOO, N MARLOWE, N BERNARD, C COCHRAN and J DILEANIS
Applied Biosystems, Foster City, CA
OBJECTIVETo evaluate the performance of HIV-1 genotyping on the new 3100 Genetic Analyzer, Applied Biosystems' 16-capillary instrument.
INTRODUCTIONThe ViroSeq™ HIV-1 Genotyping System (HGS) is a sequencing based test method (for Research Use only) for the identification of mutations in the protease and 5' end of the reverse transcriptase genes which are associated with drug resistance. This sys-tem provides all the reagents needed to prepare RNA from plasma samples, to per-form a two-step RT-PCR reaction, and to sequence the subsequent 1.8kb PCR product. In addition, the system includes the ViroSeq™ HGS Software, which will automatically assemble the sequences from a clinical research specimen into one consensus sequence (project) and perform a comparison of the consensus sequence with a refer-ence sequence. The user is then able to manually edit the data before a report is gen-erated. This report lists all the amino acid differences between the reference (HIV-1 wild type) sequence and the specimen's consensus sequence. These differences are sorted by gene (protease vs. reverse transcriptase) and sorted as variants associated with drug resistance (per the Los Alamos HIV-1 Database) or variants which are “novel” (not identified by the Los Alamos HIV-1 Database as conferring drug resis-tance).
For sequencing, the ViroSeq HIV-1 Genotyping System utilizes 6–7 sequencing primers; the use of these primers provides double coverage of the entire protease and first 315 codons of the reverse transcriptase gene. These sequencing reactions can be analyzed on a variety of instruments. To date, the 377 instrument has been the plat-form of choice for customers who require mid to high throughput HIV genotyping; this acrylamide gel-based platform permits the genotyping of up to 13 specimens per 96-lane gel with a 7 hour run. For laboratories which have a lower throughput of speci-mens, the 310 instrument can be used; this instrument is a single capillary elec-trophoresis platform that permits the genotyping of 1-2 specimens per 24 hour run. For research laboratories with very high throughput requirements, the 3700 instru-ment can be used; this 96-capillary electrophoresis instrument allows the genotyping of up to 13 specimens in 2.5 hours. User feedback suggested that a capillary instru-ment with the capabilities of the 377 would be a preferred instrument of choice since there would be no acrylamide gel and no manual loading of the sam-ples.
The 3100 instrument, an automated walk-away genetic analyzer, was released in April 2000. This instru-ment has a 16 capillary array. This platform permits the analysis of 2 HIV-1 clinical samples in 2.5 hours, using the standard sequencing mod-ule. Therefore, 20–24 samples can be analyzed within a 24 hour period.
MATERIALS AND METHODS:Randomly selected clinical research samples from 41 HIV-1 infected individuals were processed using the ViroSeq™ HIV-1 Genotyping Kit (version 2) per the manufactur-er’s protocol. For some of these samples, a second round of RT-PCR was performed with nested PCR primers. (These nested PCR primers are not included in the ViroSeq kit.) Viral loads of the samples ranged from <50 to 384,000 copies/mL. The sample preparation procedure utilized a guanidium thiocyanate lysis of the pelleted viral par-ticles. The resulting RNA was precipitated with isopropanol. After RNA resuspen-sion, a two step RT-PCR reaction was performed with the MuLV reverse transcriptase and AmpliTaq Gold® enzymes. (In the ViroSeq kit, the PCR reaction contains dUTP nucleotides and the enzyme, AmpErase® UNG (uracil N-glycosylase), for contamina-tion control.) PCR products were purified with Microcon®-100 columns and subse-quently analyzed on an agarose gel. After appropriate dilutions of the PCR products are made, the PCR products were sequenced with 7 sequencing primers using the BigDye™ terminator chemistry. These sequencing samples were analyzed with (a) a 5% Long Ranger‚ gel with the 377 DNA Sequencer and (b) POP-6™ polymer with the 3100 Genetic Analyzer (50 cm capillary array). Sequences were analyzed with Sequencing Analysis Software v.3.3 and the ViroSeq HIV-1 Genotyping Software v.2.2.
RESULTS: • Sequencing results showed concordant genotypes for sequencing reactions from the same
PCR product run on both the 3100 and the 377 instruments.
Table 1. Genotypes of Samples Analyzed on the 3100 and 377 Instruments
Sample Protease mutations Reverse Transcriptase mutations
1677 M36I, G48V, L63P, A71V, L90M M41l, M184V G196E, R211K, T215Y
1680 M36I none identified
1684 M46I, L63P, G73S V77I, N88S, L90M V179D, M184V
1679 M36I, L63P, V77I G190A, R211K
1603 M36I, G48V, V82A S68G, M184V, T215Y
1606 K20R, V77I none identified
1607 L10V, M36I, L63P R211K
1608 V77I none identified
1610 L10V, M36I, L63P V106I, R211K
1666-3 M36I
1611 A71V K70R, L74V, L100I, K103N, R211K, T215F, K219Q
1612 L63P K70R, K103N, M184V, R211K
1613 L10F, D30N, L63P, A71V, V75I, V77I, N88D M41L, M184V, T215Y
1614 L10V, M46I, G73S, V77I, L90M D67N, K70R, K103N, V108I, G190A, T215F, K219E
1615 D30N, M36I, M46I, K55R*, D60E, L63P, N88D D67N, K70R, K103R, M184V, K219Q
1617 L63P G196E*, R211K
1616 none identified R211K
1619 G16E, L63P, A71T G196E
1666-10 L63P none identified
1666-13 M36I R211K
1628 D60E, L36P, V77I R211K
1629 V77I K166R, G196E, R211K
1642 L63P, V77I V106I
• Longer read lengths were obtained with the 3100 capillary electrophoresis than with slab gel electrophoresis. On average, 700 bases could be read with the 3100 using the stan-dard StdSeq50_POP6DefaultModule with the 50 cm capillary array. The cycle time was2.5 hours for 16 sequencing reactions. (Figures 2 and 3)
Figure 2. Figure 3.
• An alternate method allows for more rapid sequencing. We tested this method with 12 specimens. When we used this rapid sequencing module (RapidSeq36_POP6DefaultModule), read length decreased to approximately 480 bases. The rapid sequencing module data collection time needs to be increased from 2100 sec-onds to 2580 seconds, which will increase the cycle time from 60 minutes to 68 minutes, if double coverage of the protease and reverse transcriptase sequences is desired. Note: Since resolution was decreased, additional edits were needed in the HGS software, there-by eliminating the advantage of the longer standard 3100 method which reduced edits by 60%. (Figures 4, 5, 6, and 7)
Model 3100
Basecaller-3100opt.bcpBC 1.2.d.4
11_primerG_F11_11.ab1
11[primerGLane 11
Signal G:480 A:461 T:544 C:268DT3100POP6{BD}v2.mob
Points 812 to 11557 Pk 1 Loc: 812
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Version 3.3
A C T G AT ATNC10
TAAT CCCT GG
20
TG T CT CATT G
30
TTTATA CTA G
40
G TAT GG TAAA
50
TG CAG TG TAC
60
TTT CTG AATT
70
CTTTAT CTAA
80
GGG AAC TG AA
90
AAATATG CA T
100
CACCCAC AT C
110
CAG TATT GTT
120
AC T GA
TTTGT
130
TC TTTTTTAA
140
CCCTGC GGG A
150
TG TGG TA TTC
160
C TAATTGAAT
170
TTCCCAG AAA
180
T CTTGAG TT C
190
TC TTATT GAG
200
TT CTC TGAAA
210
TCTAC TAA TT
220
TT CTCCAT C T
230
AGCAC T GTTC
240
TTTTTCTTT
25
A
50
T GGCAAATAC
260
T GGAG TA TT G
270
TA T GGATTTT
280
CAGGCCCAA T
290
C TTT GAAA TT
300
TTCCCTTCCT
310
TTTCCA TTT C
320
T GTACAAA TT
330
TC TATTAAT G
340
C TTTTATTTT
350
TTCTTCT GT C
360
AAT GGC CATT
370
GTTT
AAC TTT
380
T GGGC CAT CC
390
ATTCCTGG TT
400
TTAA TTTTAC
410
TGGTACAG TT
420
TCAA TA GG AC
430
TAA TGGG AAA
440
A TTTAAAG TA
450
C AG CCAA TC T
460
G AG TCAT CAA
470
ATTT CTTCCA
480
A TTAT GTT GA
490
CAGG TG TAG
GG
500
TCCTATTAAC
510
AC TG TACTTA
520
TA GC TTTAT G
530
TCCACA G ATC
540
TC TATGG CTA
550
C CTG ATCATA
560
C T G TCTTAC T
570
TTGATAAAAC
580
CTCCAATTCC
590
CCCTATTATT
600
TTT GG TTTCC
610
AT CTTCCTGG
620
TAAAT
TCCATT
630
T CTT CT AA TA
640
CT GT ATCA T C
650
TG C TCCTG TA
660
TCTAATAG AG
670
CTTCC TTT AG
680
TTG C CCC CC T
690
AT CTTTA TT G
700
T GACGAC GGG
710
T CGTTG CC AA
720
AG AGTG AT CT
730
G AGGG CAG TT
740
AAAGG ATA C T
750
T
TCT CCTTGNC760
TA TTGG CTCC
770
NG CTTCTG AG780
ANGG AGGTTG790
CTG T CTNCT C800
Model 3100
Basecaller-3100opt.bcpBC 1.2.d.4
11_primerB_B11_03.ab1
11[primerBLane 3
Signal G:840 A:1178 T:580 C:384DT3100POP6{BD}v2.mob
Points 762 to 11557 Pk 1 Loc: 762
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Version 3.3
TTTNNAAAA T10
NA AAG CAT T A20
AT A GTAAAT T
30
T G T A CAGNAA40
ATG GNAAAAG50
G AAGGG AAAA
60
T TT CAAAGNA70
TT GGG C CT G A
80
AAAT CCAT AC
90
AAT AC T C CAG
100
TA TTT GC CAT
110
AAAG AAAAAG
12
G
20
AAC AG T G C TA
130
G ATG G AG AAA
140
ATTA G TA G AT
150
TT CA G AG AAC
160
T CAA TAAG AG
170
AAC T CAAG AT
180
TTC T GGG AAA
190
TT CAAT TA G G
200
AATACCA CA T
210
C CC GC AGGG T
220
TAAAAAAG AA
230
CAAA TCA G TA
24
40
ACAA TAC T GG
250
A TGT GGGT GA
260
TGCA TA TTTT
270
TCAG TTCCC T
280
TAG ATAAAG A
290
ATTCA G AAAG
300
TA CA C TGCA T
310
TTACCAT AC C
320
T AG TA TAAAC
330
AA TG AG ACAC
340
CAGGG ATTA G
350
ATAT CA G TA C
360
AATG
GT GC TT C
370
CAC AGGG AT G
380
G AAAGG ATCA
390
C CAG CAATAT
400
TCCAAAG TAG
410
CAT GACAAAG
420
ATCCT AG AG C
430
CTTTTAG AAA
440
ACAAAAT CCA
450
G AAATGGTTA
460
T TTAT CAA TA
470
C AT GG AT GAT
480
TT GTAT GT
TAG
490
G AT CT GAC TT
500
AG AAATA GG A
510
CAGCA TA G AG
520
CAAAAATAG A
530
GGAAC TGAG A
540
CAGCAT CTG T
550
T GAGGTGGGG
560
ATTTTTCAC A
570
C CAG ACGAAA
580
AACAT CA G AA
590
A G AAC CTCCA
600
TT CCTTTGG A
610
TGGGT
TTA T GA
620
AC TCCAT CCT
630
G AT AAATGGA
640
CAGTAC AG CC
650
TAT AA T GC TG
660
CCAG AAAAAG
670
AAAG CT GG AC
680
T GT CAAT GAC
690
ATACAG AAG T
700
TAG T GGG AAA
710
A TT G AATT GG
720
GC AAGT CAG A
730
TTTA TG CAGG
74
G
40
GA TTAAA GTA
750
AAA CCAATTA
760
T G TAAA CCTT
770
N TT AG AGGAC780
CCAA AGCAC T
790
TACCGGA GGT
800
A TTA CCAC T A
810
CCAAA
Standard Rapid 3100 Sequencing Module Data
Figure 4. Figure 5.
3100 Rapid Sequencing Module Data with Additional Run Time
Figure 6. Figure 7.
• HIV-1 samples were processed using the manufacturer’s protocol. The subsequent sequencing reactions were run on both the 377 and the 3100. After HGS software analy-sis, the number of edits made in the projects with 377 data was compared with the num-ber of edits made in projects with 3100 data. The 377 projects showed a total of 5603 edits; the 3100 projects showed a total of 1743 edits. Therefore, there were approximate-ly 3X less edits needed when sequencing reactions were analyzed on the 3100 instru-ment. Therefore basecalling accuracy was improved tremendously with the 3100 data. (Figures 8 and 9)
Figure 8.
Figure 9.
0
100
200
300
400
5003773100
Number of edits
Sample
Model 3100
Basecaller-3100RRBC 1.2.d.4
B03_4-2_b_03.ab112005-171 4-2_b Lane 3
Signal G:683 A:768 T:406 C:316DT3100POP6{BD}v2.mob
Points 857 to 18378 Pk 1 Loc: 857
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Version 3.4.1
ATAAGG CATT
10
AGNTAGTAAA20
TCTG TACAGC
30
AAATGG AAAA
40
GGGAAGG AAA
50
AATTTNAAAA60
TTGGG CCTGA
70
AAATCCATAT
80
AA TACTCCG T
90
ATTTGCCATA
100
AAG AAAAAAG
110
ATAG TACTAA
120
GTGGAG
GGAAG
130
TTAGTAGATT
140
TCAGAGAACT
150
TAATAAGAGA
160
ACTCAAGACT
170
TCTGGGAAGT
180
TCAATTAGGA
190
ATACCACATC
200
CTGCAGGGTT
210
AAAGAAGAAA
220
AAATCAGTAA
230
CAGTACTAGA
240
TGTGGGTGAT
250
GCATATTT
TTT
260
CAGTTCCTTT
270
AGATAAGACT
280
TCAGAAAGTA
290
TACTGCATTT
300
ACCATCCCAG
310
TNTAACAATG320
AGACT CANGG330
ATAGATTTAG
340
TNCATGNGCT350
CCACNGGATG360
GAAGGATCNC370
NGCA TNTTCC380
AAGT
TGCATGA
390
CAAATNTTAA400
GCCNTTTAGG410
AACAAA T CCN420
ACTAGNT TTN430
NTCAAACANG440
GGGATTGGTG
450
GGGACTGATT
460
NAAATNGGCG470
C TTTGCCAAA
480
TNGGG CCGGG490
NCCCTTTTTA500
GGGG
GGG TTTC
510
CCCCCCCAAA
520
AACTTAAANA530
ACCCCTTTTT
540
GGNGGGTTAA550
GCCCCNCTGN560
AAAGGGANGG570
GCCAANCNTN580
CCNAAAAAAG590
GGGGGTTAAA
600
AAAAAAAANT610
TNGGAAAAAA620
T GGGCCAAAA
630
T
Model 3100
Basecaller-3100RRBC 1.2.d.4
A01_2-2_a_01.ab112005-171 2-2_a Lane 1
Signal G:734 A:747 T:386 C:325DT3100POP6{BD}v2.mob
Points 917 to 18378 Pk 1 Loc: 917
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Version 3.4.1
GACTTANGNT10
TTGGGGNAGN20
ATACAACAAC
30
TCCCTCT NGN40
AGNCGG AG AC50
GATAG ACAAG
60
GNAACTG TAT70
CCTTTAGNCT80
TCCCTCAAAT
90
CACTCTTTGG
100
CAACGACCCC
110
TCGTCCCAAT
120
AAGG
ATAGGG
130
GGGCAACTAA
140
AGGAAGCTCT
150
CTTAG ATACA
160
GGAGCAGATG
170
ATACAGTATT
180
AGAAGAAATA
190
GATTTGCCAG
200
GAAGATGGAA
210
ACCAAAAATG
220
ATAGTGGGAA
230
TTGGAGGTTT
240
TATCAA
AGTA
250
AGACAG TATG
260
ATCAGGTACC
270
CCTAGAAATC
280
TGTGGACATA
290
AAG TTATAGG
300
TACAGTATTA
310
GTAGGACCTA
320
CACCTGTCAA
330
CATAATTGGA
340
AGAAATCTAA
350
TGACTCAGCT
360
TGGGTGCACT
37
T
70
TTAA TTTTNC380
CATTAG TCCT
390
ATTGAACTGT
400
NCNGTAAAA T410
TAAGCCNGGA420
ATGGAT GGCC
430
CAAAGTTAAA
440
CA TGGCCATT
450
G CNGAAG AAA460
A TTAA GCNTT470
GTTG AATNTG480
TCNG
AACTGG
490
AANGAANG AA500
ATTTNAAANT510
GGGCCGAAAT
520
CCTCCAT CCT
530
CCGTTTTGC T
540
TTAAN AAAAN550
ACG TC TAATG
560
GGNAAANTTT570
TANTT CNAAA580
ACTTATAAGA
590
AACCCA
Model 3100
Basecaller-3100RR.bcpBC 1.2.d.5
2-2 3100rr[b
2-2 rr3100[bLane 3
Signal G:343 A:502 T:257 C:177DT3100POP6{BD}v2.mob
Points 2078 to 14950 Pk 1 Loc: 2078
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Version 3.4.1
AAATAA AAG C
10
ATTA G TA G AA
20
ATC T GTACAG
30
AAC T GG AAAA
40
GG AAAGG AAA
50
ATTT CAAAAA
60
TTGGG C CTG A
70
AAATCCA TA C
80
AA TAC TCCAG
90
TATTTGC TA T
100
AAAG AAAAAA
110
G ACA G TAC TA
120
AATGG
AG AAA
130
A TTA G TA G AT
140
TTCA G AG AAC
150
TTAATAA G AG
160
AACCCAAG AC
170
TTTT GGG AAG
180
TTCAA TTA GG
190
AATAC CAC AT
200
CCT GCAGGG T
210
TAAAAAAG AA
220
AAAAT CA GTA
230
A CA G TA C T GG
240
AT G T GGG TG
GA
250
T GCA TA TTTT
260
TCAG T TCC TT
270
TAG ATAAA G A
280
C TTCAGG AAG
290
TA TA C T GC AT
300
TTACCA TA CC
310
TAG TACAAA C
320
AA T GA GACAC
330
CAGGGG TTAG
340
ATAT CA G TA C
350
AAT G TAC TT C
360
CAC AAGG AT G
370
G
GAAAGGGT CA
380
C CAG C AA T AT
390
T CCAAAG T AG
400
CA T G A CAAAA
410
A T CT T A G AG C
420
C TT T TA G AA A
430
A C AA AA T C CA
440
G A C AT G G TT A
450
T C T A T C A A T A
460
C G T G G AT G A T
470
T T G T A T G T A
G
480
G AT C T G A C T T
490
A G AAA T A G A A
500
C A G C T T AG
Model 3100
Basecaller-3100RR.bcpBC 1.2.d.5
2-2 3100rr[a
2-2 rr3100[aLane 1
Signal G:482 A:550 T:333 C:221DT3100POP6{BD}v2.mob
Points 2500 to 14950 Pk 1 Loc: 2500
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Version 3.4.1
CTTT A C T CCC
10
T CTCAG AAGC
20
A GG AG AC GA T
30
AG ACAAGG AA
40
C TGTAT CCTT
50
TAG C TTCCCT
60
CAAAT CA C T C
70
TTTGGCAA C G
80
A CCCCT CG TC
90
CCAA TAAGG A
100
TAGGGGGGCA
110
AC TAAAGG AA
120
G C TC
TC TTAG
130
ATACA GG AG C
140
A G ATGATAC A
150
G TA TTAG AA G
160
AAATA G ATTT
170
GCCA GG AA GA
180
TGGAAACCAA
190
AAATGA TA G T
200
GGG AATT GG A
210
GGTTTTA TCA
220
AAG TAA GACA
230
GTAT GA TCA G
240
GTACCCC T
TAG
250
AAATC T GTGG
260
ACA TAAAG TT
270
A TA GG TAC AG
280
TA TTA G TA GG
290
AC C TACACC T
300
G TCAACA TAA
310
TT GGAA GAAA
320
TC TAA T GA C T
330
CAGC TT GGG T
340
GCAC TTTAAA
350
TTTTCCCAT T
360
AG T CCT AT TG
370
AAAC T G TACC
380
A G TAAAA TTA
390
AAG C CAG G AA
400
T G G AT GG C CC
410
AAAA G T TAA A
420
C AA T G G C C AT
430
T G A C AG AA G A
440
AAAAA T AA AA
450
G C AT T AG T AG
460
AAAA T CT G T A
470
C A G AA C T GGG
480
AA
AA G G AAA G
490
GGAAA A T
• In 16 random pairs of data, 8 samples had mixtures which were called automatically more often in the 3100 vs. 377 data; for 4 samples, the mixtures were called more fre-quently in the 377 data; and for the remaining 4 samples, mixture calling was equal. Therefore improvements in automatic base calling of mixtures was seen in the 3100 data. These improvements resulted in less time needed for manual editing. (Figures 10, 11, and 12)
Figure 10. Figure 11.
Figure 12.
CONCLUSIONS: • Both instruments, the 377 and 3100, provided double coverage sequences for the entire
HIV-1 protease gene and for the first 315 codons of the reverse transcriptase gene.
• Longer read lengths were observed when sequencing reactions were analyzed on the 3100 capillary electrophoresis instrument. These longer read lengths were the result of better peak resolution at 580 base pairs and hence fewer edits in the ViroSeq‘ HGS software were needed. This decrease in number of edits reduced the time needed to review a project.
• Longer read lengths also provide additional sequencing coverage for the targeted genes. This additional information results in a higher confidence in base calling.
• Concordant genotypes were generated from the 377 and 3100 instruments.
• In addition, use of the capillary electrophoresis instrument eliminated the need to pour an acrylamide sequencing gel and manual loading of the samples onto the acrylamide sequencing gel, thereby permitting a more automated, walk-away system, a reduction in labor cost, and increased ease of use in research laboratories.
For Research Use Only. Not for use in diagnostic procedures.
The ViroSeq™ HIV-1 Genotyping System, Version 2, is for Research Use Only. Not for use in diagnostics procedures. Under separate procedures and labeling, this type of product can be used for investigational use. Please contact Applied Biosystems for information.
ViroSeq and BigDye are trademarks and MicroAmp and Applied Biosystems are registered trademarks of PE Corporation or its subsidiaries in the U. S. and certain other countries. AmpErase is a registered trademark of Roche Molecular Systems, Inc. Microcon in a registered trademark of Millipore. All other trademarks are properties of their respective owners.
3100 data
377 data