Supplementary Information
SNP Assay Design
The sequence surrounding 8 snps, previously identified as having an impact on either
1
HIV viral load (rs2395029 and rs9264942) or time to HIV disease progression
(rs9261129, rs9261174, rs3869068, rs2301753, rs207480 and rs2074479) were
downloaded from the dbSNP database (www.ncbi.nlm.nih.gov/projects/SNP/) and
loaded into RSFormat in the My Sequenom section of the Sequenom TM website
(www.sequenom.com). MassARRAYAssay Design 3.1 software (SequenomTM, San
Diego, CA) was used to design the amplicon and extension primer assays using
iPEXGold chemistry (SequenomTM) (Table 1). Automated calling was performed by
Sequenom Typer 4.0.3. All calls were verified by manual inspection.
SNP Assay Validation
SNPs were designed and either multiplexed to run in two wells, one with 5 snps and
one with 4 SNPs, or multiplexed to run in a single well. DNA samples (N=31) from a
variety of sources (including UCLA KIR exchange samples, cell lines from the
International Histocompatibility Working Group and a few MACS samples) were run in
duplicate in the 8-plex assay and in duplicate in the 4- and 5-plex assays. Table 2
presents the genotype of samples used in validation of the assays for the 8 SNPs used
in the present study. Seven samples were randomly chosen to run all assays in
duplicate as single assays, using the same primers that were chosen for the 8-plex. All
samples were therefore run either 4 times (9-plex and 4-, 5-plex) or 6 times (9plex, 4-
and 5-plex and single assay formats). All assays were consistent in the calls,
independent of the assay multiplex level.
MACS Sample Preparation
DNA was isolated from PBMC using QIAGEN extraction columns and quantified with
SYBR Green (Invitrogen.) Institutional protocols for working with potentially infectious
material were closely adhered to during all phases of this work. Samples were
normalized in 96 well plates and 2.5 ng of each sample was transferred to 384-well
PCR plates for running the iPLEX chemistry according to SequenomTM protocols. All
MACS samples were blinded for case-control status and each 384-well plate contained
a total of 9 positive and 9 negative controls. A total of 10% of the MACS samples were
examined in duplicate as a quality control measure.
Sequencing of Samples for rs2074479 Assay Confirmation
Thirteen of the MACS samples, which were heterozygous for SNPS rs9261129,
rs9261174, rs3869068, rs2301753 and rs2074480 yielded homozygous C calls for
assay rs2074479. Because this was unexpected, Primer3 was used to design a
different set of primers flanking rs2074479. These primers, (5’aattcccttccctcccttct 3’ and
5’cagagggctgatttcctgac 3’) were used to amplify a 163 bp fragment containing the
rs2074479 SNP. These amplicons were purified on DNA Clean and Concentrator-5
columns (Zymo Research) and sequenced using Applied Biosystems BigDye 3.1
chemistry. 1ul of the purified amplicon was combined with 5pmoles of either the forward
or reverse primer, and 2ul BigDye master mix with 4ul 5x sequencing buffer, in a 20ul
reaction. Reactions were cycled according to manufactures recommendations.
Sequencing products were purified by ethanol precipitation and re-suspended in
formamide prior to loading on the ABI 3130xl capillary sequencer.
Further confirmation was obtained by FokI restriction enzyme (New England BioLabs)
digestion of the 163 bp fragment. DNA which is homozygous T at the SNP is cut twice
by the enzyme, yielding fragments of 69, 49 and 45bp. DNA that is homozygous C is cut
once, yielding fragments of 94 and 69bp; and DNA that is heterozygous, CT, at that
SNP, yields fragments of 94, 69, 49 and 45 bp. These fragments were resolved on a 4%
Metaphor agarose (Lonza Rockland, Inc.) 1X TBE gel. All samples in question were
determined to contain both the C and the T allele at the SNP site by careful examination
of the sequence chromatograms.
Table 1. Primers used for assaying SNPs on the MALDI-TOF mass spectrometer.
Upstream PCR
Downstream PCR
SNP ID
Primer
Primer
rs9261129
GAGCTGCTGTCAGTGGAGAT
TCTCCTCCATTGAATGCCTG
gttCCCTCTCCTCTGTGT
rs9261174
ATGCCAATACCTTGCTTGCC
GCTGGAAGTATCACAGTACC
TGCTTGCCATTTTGGTTAC
rs3869068
CTTCAATGCAAACCCATGAG
CTCCCTTTCCCTGTCTATAC
AAACCCATGAGAAGCCC
rs2301753
CCTCCTGCAGAGACTCTTCT
CCACTGCATAGTGGCTCTC
rs2074480
AAGTGATCAGGTCCTTTTTG
TTTCAGCCACCTACTTTGCG
aAGTTTCCTAGTACTGCTCT
rs2074479
CCCTCATCTGTGTAGATTTG
CCACTGGATAATCTTCAGGG
AGATTTGAAGTCCCAACA
rs9264942
TTGTCCCACAAGAGACAGAC
ATGAGCTTCCAGGAGCAGG
rs2395029
TTCTCACCCGCTGGTCTCT
TTTCAGCCACCTACTTTGCG
Extension Primer
ctCTCTCCTCGTCGTCC
GACAGACCCACTTCC
CTGTCCAATTCCCCTG
Upstream and downstream PCR primers were ordered with an additional 10-base tag,
ACGTTGGATG at the 5’ end, which is the standard default design for SequenomTM
Assay Design. Nucleotides designated by small letters at the 5’ end of some extension
primers are short tags added to the sequence that allow for assay mutiplexing.
Table 2. Genotype of samples used in validation of the assays for the 8 SNPs used in
the present study.
Sample
rs9261129
rs9261174
rs3869068
rs2074480
rs2074479
rs9264942
rs2395029
1
T
T
G
A
T
T
T
2
T
T
G
C
A
T
T
T
3
TC
CT
AG
CA
CA
CT
C
T
4
TC
CT
AG
CA
CA
CT
T
T
5
TC
CT
AG
CA
CA
CT
T
T
6
TC
CT
AG
CA
CA
CT
CT
T
7
T
T
G
C
A
T
T
T
8
TC
CT
AG
CA
CA
CT
CT
T
9
T
T
G
C
A
T
T
T
10
T
T
G
C
A
T
CT
T
11
T
T
G
C
A
T
CT
T
12
TC
CT
AG
CA
CA
CT
T
T
13
T
T
G
C
A
T
T
T
14
T
T
G
C
A
T
CT
T
15
TC
CT
AG
CA
CA
CT
CT
T
16
T
T
G
C
A
T
T
T
17
T
T
G
C
A
T
CT
GT
18
TC
CT
AG
CA
CA
CT
T
T
rs2301753
19
TC
CT
AG
CA
CA
CT
C
GT
20
C
C
A
A
C
C
C
T
21
T
T
G
C
A
T
CT
T
22
T
T
G
C
A
T
T
T
23
T
T
G
C
A
T
CT
T
24
C
C
A
A
C
C
T
T
25
T
T
G
C
A
T
T
T
26
TC
CT
AG
CA
CA
CT
CT
T
27
T
T
G
C
A
T
C
T
28
TC
T
AG
CA
A
CT
CT
T
29
TC
CT
AG
CA
CA
CT
T
T
30
T
T
G
C
A
T
T
T
31
T
T
G
C
A
T
CT
T
neg
null
null
null
null
null
null
null
null