Vol. 21 no.
17 2005, pages 3535–3540
BIOINFORMATICS ORIGINAL PAPER
Genetics and population analysis
A statistical model for HIV-1 sequence classification
using the subtype analyser (STAR)
R. E. Myers1 , C. V. Gale2 , A. Harrison3 , Y. Takeuchi1 and P. Kellam2,∗
1 Department of Immunology and Molecular Pathology, 2 Department of Infection and
3 Department of Biochemistry, University College, London
Received on March 23, 2005; revised on June 23, 2005; accepted on June 30, 2005
Advance Access publication July 26, 2005
ABSTRACT
Motivation: HIV-1 antiretroviral drug resistance testing produces large
amounts of HIV-1 protease and reverse transcriptase sequences.
These provide an excellent resource to study the incidence, spread
and clinical significance of HIV-1 subtypes. We have produced a pro-
gram, Subtype Analyser (STAR) that rapidly and accurately subtypes
HIV-1. Here we have determined a robust and statistically validated
model for subtype assignment.
Results: We have significantly extended our HIV-1 subtyping tool
(STAR), such that each query sequence when evaluated against sub-
type profile alignments, returns a discriminating score based on the
ratio of subtype positive to negative amino acid positions. These scores
were transformed into a Z -score distribution and evaluated. Of the 141
sequences used to define the subtype alignments, 98% were correctly
reclassified. InclusionofadditionalrecombinationdetectionwithinSTAR
increased the detection of known recombinant sequences to 95%.
Availability: STAR is available as compiled (Linux Fedora 3) or source
code from http://pgv19.virol.ucl.ac.uk/download/star_linux.tar
Contact: p.kellam@ucl.ac.uk
Supplementary information: Available at http://pgv19.virol.ucl.ac.uk/
download/star_supplement
1 INTRODUCTION
Human immunodeficiency virus type-1 (HIV-1) is the retroviral
agent that causes acquired immune deficiency syndrome (AIDS).
As of December 2004 there were an estimated 40 million people
living with HIV/AIDS, with 5 million new infections and 3 million
deaths in 2004. One defining component of the retroviral life cycle
is the reverse transcription of the viral RNA genome into DNA. The
enzyme responsible for this, reverse transcriptase (RT), has a low
level of fidelity resulting in an error rate of between 1/1000 and
1/10000 nucleotide misincorporations (Roberts et al., 1988; Takeuchi
et al., 1988). Given a 9000–10 000 base pair genome, this indicates
that progeny viruses contain between 1 and 10 polymorphisms per
genome per round of replication. HIV-1 replicates to high levels
in individual untreated patients (Ho et al., 1989), which coupled
with the size of the HIV-1 pandemic results in viral quasi-species of
HIV-1 within an infected individual and a global distribution of
viruses that are genetically divergent (Wain-Hobson, 1993).
HIV-1 genetic diversity has been stratified and a subtype nomen-
clature based on sequence variation has been described. Initial
∗ To whom correspondence should be addressed.
© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org
doi:10.1093/bioinformatics/bti569
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HIV-1 phylogenies were inferred from viral envelope sequences
(env) [subtypes A–F (Myers et al., 1992)], which were consistent
when extended to group antigen sequences (gag) [except subtype E
(Louwagie et al., 1993)]. Improvements in molecular biology, com-
putational power and phylogenetic algorithms have subsequently
allowed subtyping of the full length HIV-1 genome (Salminen et al.,
1995, 1996). Currently, there are 11 single subtypes (A–H, J, N, O)
and a variety of circulating recombinant forms (CRF) of HIV-1 that
represent recombination events between two or more single subtypes
(Robertson et al., 1995, 2000). The Los Alamos HIV-1 Sequence
Database stores updated reference subtype sequences and provides
phylogenetic tools, such as SUDI, that allow novel subtypes to be
evaluated (http://hiv-web.lanl.gov/content/hiv-db/SUDI/sudi.html).
Although HIV-1 sequences and consequently subtypes evolve,
HIV-1 subtyping allows clinical aspects of HIV-1 infection and man-
agement to be studied in the context of similar sequences. Web-based
HIV-1 subtyping tools are available at both the NCBI (Rozanov et al.,
2004) and at the Stanford HIV Drug Resistance Database http://
hivdb2.stanford.edu/asi/deployed/hiv_central.pl?program=hivseq),
both of which use reference datasets from Los Alamos. The NCBI
subtyping tool uses the BLAST algorithm along a sliding win-
dow to compare HIV nucleotide sequences with reference subtype
sequences, whereas the Stanford tool calculates a genetic distance
between the test sequence and reference sequences. Neither method
attempts to assign any confidence to the subtype prediction; both
require nucleic acid as input and neither allow batch submission of
sequences.
We have described previously the program STAR (Gale et al.,
2004), which uses a region of HIV-1 polymerase gene (Pol) [99 amino
acids of protease (PR), 340 amino acids of RT] to subtype sequences
relative to a large set of accurately defined reference sequences. This
region of HIV-1 is routinely used for drug resistance profiling thereby
allowing subtyping of clinical sequences evaluated for drug resist-
ance. STAR was designed to use amino acid sequences rather than
nucleic acid, to allow retrospective subtyping of sequences in data-
bases where information is stored as amino acid changes relative
to a known reference strain. STAR utilizes position-specific scoring
matrices (PSSMs) of each HIV subtype to perform profile subtype
alignments. The frequency of amino acids at each position in the Pol
fragment for a given subtype were compared against a sequence of
unknown subtype with the maximum sum of the frequencies identi-
fying the subtype of the unknown sequence. Any sequence with a
score within 1.5 standard deviations from the mean of the reference
3535
R.E.Myers et al.
10
Odds-ratio
1
0.1
0 50 100 150 200
Amino acid
Fig. 1. Odds ratio scores of a HIV-1 Pol query sequence against subtype B (black) and D (grey) PSSMs, plotted as log odds ratio for visualization. Positive
discriminant thresholds (PD) and negative discriminant (ND) thresholds were used to prevent incorporation of noise (scores around the neutral odds ratio of 1)
into the final score. The values of PD and ND were varied within the range 1.1–1.9 (PD) and 0.9–0.1 (ND). The ratio of the number of amino acid positions
with an odds-ratio score greater than the PD and less than the ND was calculated for each of the 11 subtype specific PSSMs. Subtypes B and D were the first
and second highest scoring PSSMs for this query sequence (DOR 12 and 0.71, respectively).
sequence subtype score was used to highlight sequences that were dif-
ficult to classify or were putative recombinant subtypes. This scoring
system was derived empirically and as such STAR required a more
accurate means of assessing how well a query sequence fitted the data
model. Here we describe an improved scoring mechanism for STAR
allowing the statistical assignment of single subtype, recombinant
subtype and unclassified subtypes.
2 SYSTEMS AND METHODS
A dataset of 174 sequences was obtained from GenBank and The Los Alamos
Database. A dataset of 174 sequences comprising full-length HIV-1 genomes
with annotated Gag, Pol and Env proteins was obtained from GenBank.
Although the number of fully annotated HIV-1 genomes continues to increase,
this dataset encompassed all single subtypes and the majority of CRFs. Of
these 174 sequences, 65 were identified as Los Alamos HIV-1 subtype refer-
ence sequences (Supplementary Table 1). A dataset of 141 sequences were
partitioned into 11 subtype alignments by assessing phylogenies derived from
sequence identity/linkage, neighbour joining and maximum parsimony meth-
ods. The 33 sequences obtained with the original dataset were defined as
recombinant or unclassified. This dataset was used to seed STAR, providing
sufficient subtype coverage and allowing complex repetitive permutations of
STAR program parameters. An additional 813 clinically derived sequences
were obtained from the Health Protection Agency (HPA) Laboratories in the
United Kingdom.
2.1 Scoring function
A multiple alignment of 141 HIV-1 Pol fragment sequences was performed
using ClustalW (version 1.8) (Higgins and Sharp, 1988; Thompson et al.,
1994). Owing to the high level of sequence conservation within HIV-1 Pol,
it was possible to generate an alignment with only two gapped positions. In
each case gaps were introduced as a result of single sequences. The posi-
tions that introduced gaps were excised from the overall alignment as they
provided no phylogenetic or subtype determining information. The 11 sub-
type defined alignments were extracted, such that each subtype alignment
corresponded with the structure of the complete 141 sequence alignment.
Subtype alignments were converted into normalized positional amino acid
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PD
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ND
250 300 350 400 450
frequency profiles and compared against a similarly normalized frequency
profile of the complete dataset. Alignment of a query sequence of unknown
subtype was performed using dynamic programming against a template
sequence derived from a complete subtype consensus sequence. This, there-
fore, represented the alignment structure of the reference datasets. In this
way the start and stop of the Pol fragment were determined, deletions in
the query sequence (uncommon) could be gapped and insertions (usually
owing to resistance mutations and therefore not subtype specific) could be
excised.
Each amino acid in the test sequence was compared with the odds ratio
(subtype/complete dataset) of that amino acid at the corresponding position
in each of the 11 subtype profile alignments. Odds ratio scores >1 indicated
a positive discriminant at that position for the given subtype alignment, <1
indicated a negative discriminant. The sums of the number of positive and
negative discriminants along the length of the sequence, expressed as a ratio
(positive discriminant/negative discriminant) yielded an overall discriminant
odds ratio (DOR) score for the test sequence against each subtype alignment.
The subtype alignment with the largest DOR score indicated the subtype that
the query sequence most closely resembled. The odds ratio scores at each
amino acid position were simply counted as positive and negative discrimin-
ants (rather than summed or multiplied) to prevent overprediction of subtype
by a few high scoring positions. To remove noise around a neutral odds ratio
score of 1, positive and negative discriminant assigning thresholds were estab-
lished (equations are detailed in Supplementary information). These meant
that only positions with an odds ratio score above a positive or below a negat-
ive discriminant threshold were counted and included the overall discriminant
ratio (Fig. 1).
To investigate the relationship between the level of positive and negative
discriminant thresholds and their effect on the DOR score, the threshold levels
were varied within ranges of 1.1–1.9/0.9–0.1 (positive/negative discriminant
threshold) (Fig. 1). The 141 sequences used to define the 11-subtype align-
ments were reclassified using these varying discriminant thresholds, with the
query sequence being removed from the subtype alignments prior to calcu-
lating the subtype odds ratio scores. Assessment of the DOR score for each
of the 141 sequences showed the average highest score from the 141 member
sequence set varied predictably; high negative discriminant (ND) and low
positive discriminant (PD) thresholds generated higher summed scores and
the converse producing lower scores, respectively (Fig. 2a).
 HIV-1 subtype analyser (STAR)

(a) (b)
0.1 0 .1 Ave. 0.1
0.1
Score
0.2 0 .2 % Mis-
0.2
24 - 27 Classified
0.3 0 .3
21 - 24 0.3
Negative Discriminant

4 -5
0.4 0 .4 18 - 21 0.4 3 - 4
0.4
15 - 18
0.5 0 .5 0.5 2 - 3
12 - 15 0.5

0.6 0 .6 1 -2
9- 1 2 0.6 0.6
6- 9 0 -1
0 .7

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0.7 0.7 0.7
3- 6
0.8 0 .8 0.8
0- 3 0.8

0.9 0 .9 0.9
0.9
1 .1 1.2 1 .3 1.4 1 .5 1.6 1 .7 1 .8 1.9 1 .1 1.2 1 .3 1.4 1 .5 1.6 1 .7 1 .8 1.9
Positive Discriminant
(c)
Z-score 2.75 Z-score 2.50 Z-score 2.25 % Above
0.1 0 .1 0.1 0 .1 0.1 Z-Score
0.2 0 .2 0.2 0 .2 0.2 0.2 9 8- 1 00
Negative Discriminant

9 6- 9 8
0.3 0 .3 0.3 0 .3 0.3 0.3
9 4- 9 6
0.4 0 .4 0.4 0 .4 0.4 0.4 9 2- 9 4

0 .5 0 .5 9 0- 9 2
0.5 0.5 0.5 0.5
8 8- 9 0
0.6 0 .6 0.6 0 .6 0.6 0.6
8 6- 8 8
0.7 0 .7 0.7 0 .7 0.7 0.7 8 4- 8 6
8 2- 8 4
0.8 0 .8 0.8 0 .8 0.8 0.8
8 0- 8 2
0.9 0 .9 0.9 0 .9 0.9 0.9
1 .1 1.2 1 .3 1.4 1 .5 1.6 1 .7 1 .8 1.9 1 .1 1.2 1 .3 1.4 1 .5 1.6 1 .7 1 .8 1.9 1 .1 1.2 1 .3 1.4 1 .5 1.6 1 .7 1 .8 1.9
Positive Discriminant

Fig. 2. (a) Mean DOR scores obtained from reclassifying the 141 sequences over a range of discriminant thresholds (1.1–1.9/0.9–0.1). The score magnitude
is colour coded from a mean high score of 24–27 (brown) to a mean low score of 0–3 (purple). Each sequence of the 141-sequence dataset was reclassified
for subtype at the given positive and negative discriminant thresholds and the average of the 141 DOR scores calculated. (b) The percentage of misclassified
sequences based on the known subtype of each of the 141-sequence dataset. 0–1% misclassification represents two misclassified sequences out of 141. (c) The
effect of positive and negative discriminant thresholds on Z-score. The percentage of sequences (n = 141), for which the highest value of an 11 point Z-score
distribution, is greater than the specified Z-score cut-offs of 2.75, 2.50 and 2.25 at varying positive and negative determinant thresholds. A standardized scale
(right) is used to indicate percentage ranging from 98–100% (brown) of maximum Z-scores being above the specified threshold to 80–82% (blue) scoring
above the threshold.

As the subtype of the 141 sequences was known, the effect of varying
discriminant thresholds could also be evaluated in terms of the number
of sequences that were erroneously classified (Fig. 2b). Classification was
adjudged incorrect if the highest scoring subtype alignment did not match the
known subtype of the sequence. The analysis showed little overall misclassi-
fication with the highest level being 4–5% misclassification (6–8 sequences).
More importantly, this analysis identified an island of discriminant threshold
values between 1.5 and 1.6 (PD) and 0.4 and 0.5 (ND) that were optimal
for classification. Using these discriminant thresholds resulted in correct
classification of all except two sequences (98.6%).
2.2 Assessment of score
Despite the identification of the optimal discriminant thresholds there was
no clear relationship between the magnitude of the DOR score from the
maximal subtype alignment and likelihood of a correct subtype reclassific-
ation (data not shown). Rather than considering the maximal subtype ratio
alone, the highest scoring subtype alignment (per query sequence) can be
considered as the extremity of the distribution of the scores generated for
the set of 11-subtype alignments. This was characterized using a Z-score
transformation resulting in distribution with a mean of 0 and a standard devi-
ation of 1 for the 11-point distribution of subtype scores (per query sequence).
The same process of reclassification of the 141 sequences following prior
removal from the subtype alignment was used to investigate Z-score trans-
formation. The maximum score per sequence from the 11 possible scores
was always >2. Analysis of Z-score frequency in terms of the percentage
of sequences above a given Z-score at varying discriminant thresholds (ana-
logous to Fig. 2a) was used to confirm the optimum discriminant threshold
levels. This established a Z-score value that maximized correct classification
of query sequence subtype (Fig. 2c).
As expected when the maximum Z-score threshold was reduced (Z-score
2.25, Fig. 2c), the overall percentage of sequences above the threshold was
increased. At the lowest Z-score (2.25) a broad range of discriminant
thresholds (PD range 1.4–1.9/ND range 0.5–0.3) resulted in >98% of
sequences being above the Z-score threshold. At a Z-score of 2.5, only
one pair of discriminant thresholds (PD 1.6/ND 0.5) resulted in >98% of
sequences with Z-scores higher than the threshold. This threshold com-
bination also correlated with the group of discriminant thresholds identified
previously as resulting in only two erroneous subtype predictions (Fig. 2b).
Again, as expected, a high Z-score threshold of 2.75 reduced the maximum

3537
R.E.Myers et al.
70
60
50
% Frequency
40
30
20
10
0
-2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3 3.5
Z-Score
Fig. 3. The frequency distribution of Z-score from the maximal subtype
alignments, (solid line) n = 141 (141 × 1) and non-maximal subtype
alignments (dashed line) n = 1410 (141 × 10). Data were generated using
STAR-s to reclassify the 141 sequences used to define the 11 subtype PSSMs.
percentage of sequences scoring above the threshold to 90–92%. In effect,
a high Z-score threshold should have low coverage but high accuracy of
subtype assignment with the converse being true of low Z-score thresholds.
Through this analysis we have identified discriminant thresholds of 1.6
(positive) and 0.5 (negative) that resulted in 139/141 sequences, matching
the subtype alignment to which they had been assigned, with a maximal
Z-score >2.5.
We investigated the distribution of the entire set of Z-scores for all query
sequences (n = 1551) partitioning them into two sets, the top Z-score set
(n = 141) and the remaining Z-score set (n = 10) (Fig. 3).
This analysis showed that there was excellent separation between the
maximal Z-score (subtype predictive alignment) and the 10 other Z-scores
(non-subtype predictive alignments). The two sequences that were not sub-
typed correctly produced maximal Z-scores <2.5. In these cases, the second
highest scoring subtype alignment Z-scores were also relatively large (1.11,
1.51). This contrasts with the remaining 139 correctly assigned sequences
with Z-scores >2.5, where the second highest scoring subtype PSSM scored
<1. The Z-score based implementation of STAR (STAR-s) performed better
than the original scoring threshold function of STAR (Gale et al., 2004). The
number of sequences scoring above the respective thresholds increased from
93% with STAR (Gale et al., 2004) to 98% with STAR-s described here.
2.3 Assessment of recombinant or unclassified
sequences
In cases where a query sequence returned a maximal Z-score below the
threshold of 2.5, the subtype of that sequence could not be rigorously assigned.
Unassigned query sequences were likely to be either recombinant sequences
that contained regions of sequence similarity to more than one subtype
alignment or sequences containing polymorphisms not defined within the
subtype alignments. In cases where sequences previously defined within the
Los Alamos database as recombinant [http://hiv-web.lanl.gov/content/index,
(Robertson et al., 2000)] were analysed relative to single subtype sequences
it was clear that, recombinant sequences generally produced low Z-scores
(Fig. 4).
Two (of three) circulating recombinant forms with subtype B and subtype F
recombinant regions (CRF12 BF) produced Z-scores >2.5; however, these
sequences predominantly contain subtype B sequence with minimal subtype F
recombinant regions (Carr et al., 2001). Consistent with the score distribution,
when 814 clinical sequences were subtyped using the Z-score function (data
not shown), the distribution of subtype B predicted sequences (maximum
Z-score, subtype B PSSM) showed greater dispersion than the reference
sequences used to define the subtype B alignment (Fig. 4).
3538
3.5
3
2.5
2
Z-Score
1.5
1
0.5
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4 0
-0.5
-1
A B C
Fig. 4. Distributions of subtype B Z-scores. Three groups of sequences are
displayed; sequences used to define the subtype B alignment and PSSM
(A, closed circle), known recombinant sequences containing subtype B (B,
open square) and clinical subtype B predicted sequences, i.e. those sequences
where the maximal Z-score corresponded with the subtype B PSSM (C, ×).
Two recombinant sequences (open square) circulating recombinant form 12
(CRF 12, subtypes B and F) have Z-scores >2.5. Only 8.4% of clinical
sequences predicted to be subtype B have Z-scores <2.5.
Nevertheless, only 8.4% (n = 56) of clinical sequences predicted to be
subtype B fall below the 2.5 Z-score threshold. This 8.4% of clinical
sequences may contain polymorphisms that are not present within our initial
subtype specific alignments or may suggest the presence of recombinant
genomes.
To identify the recombinant genomes, all query sequences were reanalysed
along the length of the sequence. Each query sequence was compared with the
11 HIV-1 subtype specific PSSMs; however the PSSMs were recalculated to
give normalized percentage occurrence of each amino acid per position within
the individual subtype alignments. The percentage occurrence of the amino
acids present in the query sequence relative to a subtype PSSM were averaged
within a sliding window of 50 and a step value of one amino acid. The subtype
initially predicted for the query sequence provided a baseline score of 0 and
the scores derived from the other 10 subtype PSSMs were compared with
that baseline. The output of this analysis showed positions within the query
sequence that were more similar to a different subtype rather than the predicted
subtype. Summing the regions of each subtype trace from the first instance
of a positive score to the first instance of a negative score (i.e. where the
similarity to the alternative PSSM finishes), highlighted regions of the query
sequence that were more similar to a subtype other than the predicted subtype
(Fig. 5).
The recombinant regions within a query sequence were formally assessed
by analysing their predicted length (if >50 amino acids) and the cumulative
total percentage sequence identity difference relative to the initial predicted
subtype. A ratio of summed percentage identity to sequence length was
required to be >1 for the region to be considered recombinant. Assessment
of the validity of mosaic recombinant architectures proved to be effective for
simple dual subtype mosaic architectures (Fig. 5).
The main aim of recombination characterization was to separate reliable
single subtype predictions from sequences which were less reliably subtyped.
Therefore, query sequences were initially subtyped using the Z-score sub-
typing program and then reanalysed to detect the presence of recombination
events. In the event of this second analysis phase detecting recombination,
the initial Z-score was assigned a null value and the query sequence was
reported as a recombinant with no statistical score.
 HIV-1 subtype analyser (STAR)

120 (a) 100

Subtype FK
Accumulative % Identity

100 Subtype A 90
80

% Sequence Classification
80
70
60
60
40
50
20 40
0 30
0 50 100 150 200 250 300 350 400 450 20
Amino Acid Position

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10
0
Fig. 5. Recombination analysis of CRF 12 (subtypes B and F). HIV–1 poly- STAR STAR-s STAR-rec
merase amino acids 1-99 PR, 1-340 RT (labelled 100–439) are indicated. The
(b) 1.00
initial subtype prediction for CRF12 was subtype B; therefore, amino acid
positions with a y-axis score of 0 are indicative of subtype B. Regions scoring
>0, amino acids 1–100, 150–195, show more similarity to the subtype FK 0.80
than B PSSMs. This analysis illustrates that the query sequence should be

classified as a recombinant sequence rather than single subtype B sequence
as initially predicted.
A comparison of three implementations of STAR, the original STAR
(STAR), the Z-score STAR (STAR-s), and the Z-score and recombination
analysis STAR (STAR-rec) (Fig. 6a) showed that STAR-rec performed bet-
ter than the other methods. STAR and STAR-rec correctly reclassify similar
numbers of single subtype sequences when the method dependent confidence
thresholds were applied (93 and 92% of 141 sequences, respectively). As
described, STAR-s performs better during this reclassification (98%); how-
ever, both STAR-s and STAR potentially classify known recombinants as a
single subtype. From 20 known recombinant sequences STAR and STAR-
s assigned 13 and 8 sequences, respectively to a single subtype, scoring
above the relevant threshold. However, STAR-rec identifies the majority
of the known recombinant sequences (19 out of 20), although not signific-
antly affecting the percentage reclassified known single subtype sequences
(Fig. 6a). The receiver operated characteristic (ROC) (Fig. 6b) formally
showed that STAR-rec had higher sensitivity and specificity that either
STAR-s or the original STAR methods.
In summary, STAR-rec, combining Z-scoring and recombination analysis,
provides a validated statistical framework for the accurate assignment of
single HIV-1 subtypes while identifying potential recombinant sequences.
3 DISCUSSION
We have described here a validated scoring system for assigning
HIV-1 sequence subtype to PR and RT sequences using the STAR
program. This scoring system works effectively on HIV-1 sequences
generated as a part of routine antiretroviral drug therapy monitoring.
The scoring algorithm has been parameterized, such that each posi-
tion in the test sequence was evaluated as being positively, negatively
or neutrally associated with a given subtype alignment expressed as
a PSSM. Optimal DOR score used to establish the association were
determined, which coupled with an additional recombination ana-
lysis gave optimal performance in terms of predictive classification
power, while minimizing false predictions. Hidden Markov models
(HMM) were considered as an alternative to PSSMs; however, the
hidden layers, use of a matched versus random score model and diffi-
culty of detecting recombinant sequences within our implementation,
rendered them less attractive than our PSSM model (addressed within
Supplementary data, HMM).
STAR has been designed to allow rapid, accurate and user-friendly
subtyping of HIV-1 sequences generated in a clinical context. It is our
Sensitivity
0.60
ST AR
STAR
0.40 ST AR-s
STAR-s
ST AR-rec
STAR-rec
0.20
0.00
0.00 0.20 0.40 0.60 0.80 1.00
1-Specificity
Fig. 6. (a) Comparison between STAR HIV-1 Pol (PR 1–99, RT 1–340)
subtyping methodologies. Methods were evaluated by comparing the num-
ber of sequences used to define subtype alignments correctly reclassified and
scoring above a threshold by the leave-one-out analysis (% Reclassification
above threshold—white bars) (n = 141). STAR methods were also evaluated
on the basis of correct identification of recombinant sequences as not belong-
ing to a single subtype (% Recombinant detection—grey bars) (n = 20). (b)
ROC analysis of three implementations of STAR. True positives (TP) were
calculated as correct subtype predictions above a Z-score threshold, False
negatives (FN), as below Z-score threshold for 141 single subtype sequences.
False positives (FP) and True negatives (TN) were assigned on the basis
of single subtype prediction for the set of known recombinant sequences
(n = 20) scoring above and below a Z-score threshold, respectively.
Sensitivity was calculated as TP/(TP+FN), Specificity was calculated as
TN/(TN+FP).
aim that clinical databases used to manage HIV-1 infected patients
could incorporate subtype information produced from the sequences
already used to monitor HIV-1 antiretroviral therapy resistance. In
this context, STAR has advantages over currently existing subtyp-
ing methodologies. STAR does not require each query sequence to
be submitted to a web-based subtyping tool and does not require
the building or interpretation of phylogenetic trees. The use of
amino acid based subtyping will allow retrospective subtyping of
sequences already stored in databases as mutations from a subtype
B consensus, which in some cases is the only sequence information
available. Importantly, STAR also gives a statistical assessment of
the accuracy of its subtype prediction.
The design of STAR as a clinical tool results in subtyping a relat-
ively small fragment of the HIV-1 genome (1317 of the 9000 base
pairs). Recombination events outside the Pol protein will not be

3539
R.E.Myers et al.
detected. Recombination events at the 5 or 3 ends of the HIV-1
PR and RT fragment, resulting in a short region of one subtype and a
longer region of a different subtype, also remain problematic. One of
the three previously identified recombinant sequences CRF12 (B/F)
was classified as subtype B with a Z-score >2.5, owing to the short
nature of the F sequence in the region of HIV-1 examined. The
remaining subtype recombinant sequences included in this analysis
all generated a highest scoring subtype alignment below Z-score
2.5. It is envisaged that relative overprediction of sequences that
appear recombinant is preferable to inaccurate prediction of genu-
inely recombinant sequences as confidently belonging to a single
HIV-1 subtype. This will provide clean datasets of accurately sub-
typed sequences and a set of putative recombinant query sequences
that will require subsequent downstream analysis by more sophist-
icated recombination analysis tools, such as LOHA (Bartosch et al.,
2004) or RDP (Martin et al., 2005).
STAR was designed to partition HIV-1 polymerase amino acid
sequences into subtypes defined by sequence variation, which com-
plements, but does not replace HIV-1 phylogenetic analyses. This
methodology is applicable to other datasets, which have been sub-
grouped by sequence variation. Full-length Hepatitis B virus (HBV)
and HIV-1 nucleotide genomes have both been subtyped using
STAR (unpublished data).
STAR is a tool that allows sutyping of a specific HIV-1 sequence
resource (drug resistance monitoring) and as an aid to investigating
the clinical implications of HIV-1 subtype. The majority of antiret-
roviral drug design and evaluation is undertaken using the HIV-1
subtype B virus, which predominates in both the US and western
European epidemics. The influx of non-subtype B HIV-1 infected
individuals into western Europe (Parry et al., 2001; Gale et al.,
2004) and the increasing availability of therapy in Africa and Asia
will generate large amounts of non-subtype B sequence data as a
part of routine drug resistance screening. Accurate subtype predic-
tion of this resource should, therefore, provide insights into the role
of HIV-1 subtype and sequence variation, with respect to the clin-
ical management and disease progression of non-subtype B infected
individuals. Incorporating subtype information into HIV-1 patient
management strategies will allow analysis of any potential subtype
related differences in response to drug therapy, transmission and
disease progression.
3540
ACKNOWLEDGEMENTS
This work was funded by the Medical Research Council (MRC)
(R.E.M.) and the Special Hospital Trustees of the Middlesex Hospital
London (C.V.G.).
Conflict of Interest: none declared.
REFERENCES
Bartosch,B. et al. (2004) Evidence and consequence of porcine endogenous retrovirus
recombination. J. Virol., 78, 13880–13890.
Downloaded from http://bioinformatics.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on June 26, 2015
Carr,J.K. et al. (2001) Diverse BF recombinants have spread widely since the introduc-
tion of HIV-1 into South America. AIDS, 15, F41–F47.
Gale,C.V. et al. (2004) Development of a novel human immunodeficiency virus type 1
subtyping tool, Subtype Analyzer (STAR): analysis of subtype distribution in
London. AIDS Res. Hum. Retroviruses, 20, 457–464.
Higgins,D.G. and Sharp,P.M. (1988) CLUSTAL: a package for performing multiple
sequence alignment on a microcomputer. Gene, 73, 237–44.
Ho,D.D. et al. (1989) Quantitation of human immunodeficiency virus type 1 in the blood
of infected persons. N. Engl. J. Med., 321, 1621–1625.
Louwagie,J. et al. (1993) Phylogenetic analysis of gag genes from 70 international
HIV-1 isolates provides evidence for multiple genotypes. AIDS, 7, 769–780.
Martin,D.P. et al. (2005) RDP2: recombination detection and analysis from sequence
alignments. Bioinformatics, 21, 260–262.
Myers,G. et al. (1992) The emergence of simian/human immunodeficiency viruses. AIDS
Res. Hum. Retroviruses, 8, 373–386.
Parry,J.V. et al. (2001) National surveillance of HIV-1 subtypes for England and Wales:
design, methods, and initial findings. J. Acquir. Immune. Defic. Syndr., 26, 381–388.
Roberts,J.D. et al. (1988) The accuracy of reverse transcriptase from HIV-1. Science
242, 1171–1173.
Robertson,D.L. et al. (2000) HIV-1 nomenclature proposal. Science, 288, 55–56.
Robertson,D.L. et al. (1995) Recombination in AIDS viruses. J. Mol. Evol., 40, 249–259.
Rozanov,M. et al. (2004) A web-based genotyping resource for viral sequences. Nucleic
Acids Res., 32(Web Server issue), W654–W659.
Salminen,M.O. et al. (1996) Full-length sequence of an ethiopian human immuno-
deficiency virus type 1 (HIV-1) isolate of genetic subtype C. AIDS Res. Hum.
Retroviruses, 12, 1329–1339.
Salminen,M.O. et al. (1995) Recovery of virtually full-length HIV-1 provirus of diverse
subtypes from primary virus cultures using the polymerase chain reaction. Virology,
213, 80–86.
Takeuchi,Y. et al. (1988) Low fidelity of cell-free DNA synthesis by reverse transcriptase
of human immunodeficiency virus. J. Virol., 62, 3900–3902.
Thompson,J.D. et al. (1994) CLUSTAL W: improving the sensitivity of progressive
multiple sequence alignment through sequence weighting, position-specific gap
penalties and weight matrix choice. Nucleic Acids Res., 22, 4673–4680.
Wain-Hobson,S. (1993) The fastest genome evolution ever described: HIV variation in
situ. Curr. Opin. Genet. Dev., 3, 878–883.