HapMap assay-design protocols

Protocols for HapMap assay-design

Affymetrix platform (used by Broad)

Defined protocols:

LSID:  urn:LSID:affymetrix.hapmap.org:Protocol:affy_assay_design_1:1

Title: Genotyping using Affymetrix arrays

Description: Genome Complexity Reduction

Sample DNAs should not be highly degraded nor contain PCR inhibitors, such as high concentrations of heme or chelating agents. For each individual assayed, 250 ng of genomic DNA are digested separately with 10 U of XbaI or HindIII (New England BioLabs) in volumes of 20 µL for 2 hours at 37 ℃. Following heat inactivation at 70 ℃ for 20 minutes, 0.25 µM of XbaI adaptor (5’-ATT ATG AGC ACG ACA GAC GCC TGA TCT-3’ and 5’phosphate –CTA GAG ATC AGG CGT CTG TCG TGC TCA TAA-3’)(Affymetrix), or HindIII adaptor (5’-ATT ATG AGC ACG ACA GAC GCC TGA TCT-3’ and 5’phosphate –AGC TAG ATC AGG CGT CTG TCG TGC TCA TAA-3’)

(Affymetrix) are ligated to the digested DNAs with T4 DNA Ligase (New England BioLabs) in 25 µL for 2 hours at 16 ℃. The ligations are stopped by heating to 70 ℃ for 20 minutes, and then diluted 4- fold with water. For each ligation reaction, two to three

PCRs are run in order to generate > 40 µg of PCR products. Each PCR contains 10 µL of the diluted ligation reactions (25 ng of starting DNA) in 100 µL volumes containing 1.0 µM of primer (5’-ATT ATG AGC ACG ACA GAC GCC TGA TCT-3’), 0.30 mM dNTPs, 1.0 mM MgSO4, 5 U Platinum® Pfx Polymerase (Invitrogen), PCR Enhancer(Invitrogen) and Pfx Amplification Buffer (Invitrogen). 30 cycles of PCRs are run with the following cycling program: 94 ℃ denaturation for 15 seconds, 60 ℃ annealing for 30 seconds, and 68 ℃ extension for 60 seconds. As a check, 3 µL of PCR products are visualized on 2% TBE agarose gels to confirm the size range of amplicons. The PCR products are purified over MinElute 96 UF PCR Purification plates (Qiagen), and recovered in 40 µL of EB buffer (Qiagen). PCR yields are measured by absorbance readings at 260 nm, and adjusted to a concentration of 40 µg per 45 µl. To allow efficient hybridization to 25-mer oligonucleotide probes, the PCR products are fragmented to < 100 bp with DNAse I. 0.20 U of DNAse I (Affymetrix) is added to 40 μg of purified PCR amplicons in a 55 µL volume containing Fragmentation Buffer (Affymetrix) for 35 minutes at 37 ℃, followed by heat inactivation at 95 ℃ for 15 minutes. Fragmentation products are visualized on 4% TBE agarose gels. The 3’ ends of the fragmented amplicons are biotinlyated by adding 214 µM of a proprietary DNA labeling reagent (Affymetrix) using Terminal Deoxynucleotidyl Transferase (Affymetrix) in 70 µL volumes for 2 hours at 37 ℃, followed by heat inactivation at 95 ℃ for 15 minutes.

Allele Specific Hybridization to Oligonucleotide Arrays The fragmented and biotinylated PCR amplicons are combined with 11.5 µg/mL human Cot-1 (Invitrogen) and 115 µg/mL herring sperm (Promega) DNAs. The DNAs are added to a hybridization solution containing 2.69 M tetramethylamonium chloride (TMACl), 5.77 mM EDTA, 56 mM MES, 5 % DMSO, 2.5 X Denhardt’s solution, and 0.0115% Tween-20 in a final volume of 260 µL. The hybridization solution was heated to 95 ℃ for 10 minutes then placed on ice. After warming to 48 ℃ for 2 minutes, 200 µL of the hybridization solution is injected into cartridges housing the oligonucleotide arrays (Affymetrix GeneChip® 100K Mapping Set: 50K Array Xba 240 and 50K Array Hind 240). Hybridizations are carried out at 48 ℃ for 16 to 18 hours in a rotisserie rotating at 60 rpm. Following the overnight hybridization, the arrays are washed with 6X SSPE and 0.01% Tween-20 at 25 ℃, then more stringently washed with 0.6X SSPE and 0.01% Tween-20 at 45 ℃. Hybridization signals are generated in a three step signal amplification process: 10µg/mL streptavidin R-phycoerythrin (SAPE) conjugate (Molecular Probes) is added to the biotinylated targets hybridized to the oligonucleotide probes, and washed with 6X SSPE and 0.01% Tween-20 at 25 ℃; followed by the addition of 5µg/mL biotinylated goat anti-streptavidin (Vector) to increase the effective number of biotin molecules on the target; and finally SAPE is added once again and washed extensively with 6X SSPE and 0.01% Tween-20 at 30 ℃.

 The SAPE and antibody were added to arrays in 6X SSPE, 1X Denhardt’s solution and 0.01% Tween-20at 25 ℃ for 10 minutes each. Following the final wash, the arrays are kept in Holding buffer (100mM MES, 1M [Na+], 0.01% Tween-20). The washing and staining procedures are run on Affymetrix fluidics stations. Arrays are scanned using GCS3000 scanners with AutoLoaders (Affymetrix). Scan images are processed to get hybridization signal intensity values using GCOS 2.0 software (Affymetrix). The DM genotype calling algorithm is implemented in GenoTyping Tools (GTT) (Affymetrix) and GDAS 3.0 (Affymetrix) analysis software.from Hajime Matsuzaki, Shoulian Dong, Halina Loi, Xiaojun Di, Guoying Liu,Earl Hubbell, Jane Law, Tam Berntsen, Monica Chadha, Henry Hui, Geoffrey Yang, Giulia C Kennedy, Teresa A Webster, Simon Cawley, P Sean Walsh, Keith W Jones, Stephen P A Fodor & Rui Mei. Genotyping over 100,000 SNPs on a pair of oligonucleotide arrays. Nature Methods  1, 109 - 111 (2004) . PubMed ID: 15782172)

BeadArray platform (used by Broad, Illumina, Sanger, McGill, Beijing, Shanghai/Taipei)

Defined protocols:

LSID:  urn:LSID:illumina.hapmap.org:Protocol:Golden_Gate_assay_design_1.0.0:1

Title: GoldenGateTM Assay Design Protocol

Description:

Candidate SNPs first undergo an informatic screen for suitability for assay design.  SNPs can be excluded from assay design if they are located near or within palindromic sequences, GT- or AT-rich regions or repeats similar to sequences elsewhere in the genome.

Three assay oligos are designed for each SNP locus that passes screening.  There are two 5' assay oligos, designed to hybridize upstream of the SNP, with the 3í base of each being allele-specific. The third oligo is located 3' of the two allele-specific oligos (ASOs), and is a locus-specific oligo (LSO).  All three oligonucleotide sequences also contain universal PCR primer sites. 

The LSO contains a unique address sequence that is complementary to a capture sequence on the array.

LSID:  urn:LSID:illumina.hapmap.org:Protocol:Golden_Gate_assay_design_1.1.0:1

Title: GoldenGateTM Assay Design Protocol

Description: Candidate SNPs first undergo an informatic screen for suitability for assay design.  SNPs can be excluded from assay design if they are located near or within palindromic sequences,

GT- or AT-rich regions or repeats similar to sequences elsewhere in the genome.

Three assay oligos are designed for each SNP locus that passes screening.  There are two 5' assay oligos, designed to hybridize upstream of the SNP, with the 3í base of each being allele-specific. The third oligo is located 3' of the two allele-specific oligos (ASOs), and is a locus-specific oligo (LSO).  All three oligonucleotide sequences also contain universal PCR primer sites.  The LSO contains a unique address sequence that is complementary to a capture sequence on the array.

FP-TDI platform (used by UCSF/WashU)

Defined protocols:

LSID:  urn:lsid:ucsf-wu.hapmap.org:Protocol:assay_design:1

Title: Genotyping Assay Design for Single Base Extention and FP-TDI Detection

Description:

For each SNP two PCR primers and a forward and reverse single base extention SNP  primer design was attempted. After obtainign uniquely mapped SNPs with other known SNPs marked in the sequence the design was attempted in two different steps: 1) SNP  primer design and 2) PCR primer design.

1) SNP Primer Design

The TM of the shortest possible (14 bases) was calculated using Nearest Neighbor  Theromodynamics (Beasley, E.M., R.M. Myers, D.R. Cox and L.C. Lazzeroni. 1999.

Statistical Refinement of Primer Design Parameters., pp. 55-71. In D.H.G. Michael A. 

Innis, John J. Sninsky ). If the primer TM was less than 50 degrees C the primer was  extended another base until the TM was between 50-55 degrees (using Allawi and  SantaLucia Nearest Neighbor Sequence Dependent Thermodynamic Parameters as described in Owczarzy, R., Vallone, P.M., Gallo, F.J., Paner, T.M., Lane, M.J. 1997.

Predicting Sequence-Dependent Melting Stability of Short Duplex DNA Oligomers.

Biopoly 44: 217-239.) or until the length of the primer as greater than 40 bases.

If a primer between 14-40 bases long with a TM of 50-55 degrees was found for  both the forward and reverse SNP primers a preferred SNP primer was chosen to try exprimentally by assigning penalties according to the criteria in Table 1. The penalty  scores are then compared. If the primers have different penalty scores pick the one  with the lowest. If the penalty scores are the same and the alleles are C/T or G/A pick the primer that will result in genotyping C/T (best for FP-TDI), other wise pick the shortest  primer (cheapest). If a primer is still not picked default to picking forward primer.

Invader platform (used by RIKEN)

Defined protocols:

LSID:  urn:LSID:imsut-riken.hapmap.org:Protocol:assay_design:1

Title: Design of the Invader Genotyping Assay

Description:

For each SNP two PCR primers and Invader Genotyping Assay was attempted.

After obtaining uniquely mapped SNPs with other known SNPs marked in the sequence,

the design was attempted in two different steps: 1) PCR Primer Design and 2) Invader Genotyping Assay design.

1) PCR Primer Design

Following Repeat Masking of sequence, PCR Primer were designed using Primer3 release 0.9(Steve Rozen, Helen J. Skaletsky (1996, 1997, 1998) Primer3. Code available at http://www-genome.wi.mit.edu/genome_software/other/primer3.html) with similar parameters as described previously (Vieux, E.F., Kwok, P.Y., Miller, R.D. Primer design for PCR and sequencing in high-throughput analysis of SNPs. Biotechniques 32:S28-S32, with the minor changes:

TARGET=SNP_Position-23 bases, 47 bases

       PRIMER_PRODUCT_SIZE_RANGE=81-600

       PRIMER_FIRST_BASE_INDEX=1

       PRIMER_GC_CLAMP=1

       PRIMER_OPT_SIZE=20

       PRIMER_MAX_SIZE=25

       PRIMER_MIN_SIZE=17

       PRIMER_OPT_TM=60

       PRIMER_MAX_TM=64

       PRIMER_MIN_TM=58

       PRIMER_MAX_DIFF=100

       PRIMER_MIN_GC=30

       PRIMER_MAX_GC=70

       PRIMER_MAX_POLY=3

       PRIMER_NUM_RETURN=1000

2) Invader Assay Design

1. To design an Invader assay for SNP genotyping, the sequence of 40-50 bases on each side of the polymorphic site on the target must be known. Although either the sense or antisense DNA strand can be used, certain features of the probes, such as four or more Gs in a row or sequences that might cause the target-specific region of the primary (signal) probe to form a secondary structure with its 5' flap region, indicate that the opposite target strand should be used instead.

2. Primary probes used in the Invader assay have a 5' flap and a target-specific region. The base at the SNP site on the target DNA determines the base at the 5' end of the target-specific region. In addition, the length of the target-specific region is chosen so that the Tm of the probe-target duplex is approximately 63 degrees C. The Tm can be calculated with the Hyther program developed by Peyret and SantaLucia at Wayne State University (http://jsl1.chem.wayne.edu/Hyther/hythermenu.html) or by any similar program using nearest-neighbor parameters for DNA (11,12) and including the concentrations of the probe 1uM. Because the target-specific region of each primary probe will detect only one polymorphic nucleotide at the SNP site, two unique target-specific regions must be designed for a typical di-allelic SNP locus.  To complete the primary probe design, the target-specific region is extended at the 5' end with one of the universal 5' flap sequences.

These universal 5' flap sequences are independent from the target sequence. As a result, practically any SNP assay can use primary probes designed with different target-specific regions, but the identical two 5' flap sequences. .

3. The design of the invasive probe starts with its 3' terminal nucleotide. That nucleotide overlaps with the primary probe's target-specific region at the SNP site and should be non-complementary to the polymorphic nucleotides at the SNP site, following

the order T = C > A > G. Because of this design feature, the identical invasive probe can be used with both primary probes for a particular target. Except for its 3' terminal nucleotide, the invasive probe is complementary to the target. The length of the invasiveprobe is chosen so that the Tm of the probe-target duplex is approximately of 73-78 degrees C or 10-15 degrees C higher than that of the primary probe.

4.The two FRET cassettes complementing the 5' flaps of the primary probes complete the design of the Invader assay. Like the 5' flaps, the two FRET cassettes are designed to be universal; the identical FRET cassettes can be used successfully in practically any Invader reaction. Both probes use fluorescent dyes FAM and VIC in combination with a quencher (dabcyl-dT).  The 3' ends of the FRET probes are blocked with an amino group (Glen Research).

Reference:

Single Nucleotide Polymorphisms (Methods and Protocols) Volume 212,  Chapter 16,

V. Lyamichev and B. Neri pp.229-240 Humana Press.2002.

MIP platform (used by Baylor)

Defined protocols:

LSID:  urn:LSID:bcm.hapmap.org:Protocol:assay-design_0001:1

Title: Assay Design for MIP Genotyping

Description:

For each SNP a single MIP probe is designed. A minimum of 5000 probes are designed as a probe set.

1) MIP probe design

Two homologies are designed that abut, but do not overlap the SNP. The tag sequence, two primer sequences and a restriction sequence are added to homology 1 and homology 2 in the order (5' - 3') homology 2, tag sequence, restriction site, primer P2, primer P1, and homology 1.

Perlegen platform (used by Perlegen)

Defined protocols:

LSID:  urn:lsid:perlegen.hapmap.org:Protocol:LR_design_1.0.0:1

Title: Perlegen Assay Design Protocol

Description: Long-range PCR assays were designed using Oligo primer design software(Molecular Biology Insights).  Primers were selected to have similar stringency and to map uniquely to NCBI Build 33.  From a collection of all suitable candidate primers, we used custom software to select a minimum spanning set having maximum coverage with minimal overlap between adjacent amplicons.

Genotyping arrays of 25-bp oligonucleotides were designed as four sets of 20 features (80 features per SNP), corresponding to forward and reverse strand tilings of sequences complementary to each of two SNP alleles.  A set of 20 features consisted of five sets of 4 features where the location of the SNP within the oligonucleotide varies from position 11 to position 15.  A set of four features consisted of sequences where A, C, T, or G is substituted at position 13.  Thus,each set of four features provided one perfect match to the sequence of the corresponding SNP allele and three features with a single-base mismatch for that allele.  Mismatch probes were used to measure background, and by comparison with the signal for the perfect match probes, to detect the presence or absence of a specific PCR product.

Sequenom platform (used by Broad, Beijing, HongKong)

Defined protocols:

LSID:  urn:lsid:wicgr.hapmap.org:Protocol:assay_design_1:1

Title: Primer and probe design with Sequenom SpectroDESIGNER

Description:     All genotyping reactions were performed in multiplex format (maximunm 7-fold, minimum 4-fold,average 6.5-fold).  For each SNP, a forward PCR primer, a reverse PCR primer and a primer extension probe were designed. SNPs were assorted into multiplex groups and constituent PCR primer and probes were designed using SpectroDESIGNER software version. 2.0 (Sequenom, San Diego, CA). The assay design filter for SNP sequences is 2 alleles.  The length of the amplicon (Bp) for SNP capture min 60 opt 90 max 120 with the hME-10 Primary and Secondary tags.  Extend primer design length is 17-24 (Bp) with a temperature of 45-100 oC.  Peak masses are seperated by a min 42Da with an upper limit at 8250Da.