Protocols for HapMap genotyping
Affymetrix platform (used by Broad)
Defined protocols:
LSID: urn:LSID:affymetrix.hapmap.org:Protocol:genotype_protocol_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 °C. Following heat inactivation at 70 °C
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 °C. The ligations are stopped by heating to 70 °C 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 °C denaturation for 15 seconds, 60 °C annealing for
30 seconds, and 68 °C 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
ug of purified PCR amplicons in a 55 µL volume containing Fragmentation Buffer
(Affymetrix) for 35 minutes at 37 °C, followed by heat inactivation at 95 °C 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 °C, followed by heat inactivation at 95 °C 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 °C for 10 minutes then placed on ice. After warming to 48 °C 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 °C 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 °C, then more stringently washed with 0.6X SSPE and
0.01% Tween-20 at 45 °C. 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 °C; 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 °C. The SAPE and
antibody were added to arrays in 6X SSPE, 1X Denhardt’s solution and 0.01% Tween-20
at 25 °C 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_1.0.0:1
Title: The Illumina Genotyping Facility
Description:
Shen R, Rubano T, Fan JB, Oliphant A. ìOptimizing Production-Scale Genotyping. Assay
Tutorial: High-Multiplex SNP Genotyping Assay Benefits from Integration with Turnkey
Production System. Genetic Engineering News 23(6), 2003.
The Illumina Genotyping Facility is based on an informatically integrated production process.
Standard off-the-shelf robotics and laboratory equipment are combined with Illuminaís Golden
GateTM assay, SentrixTM array matrices and SherlockTM scanner technology to form a highly
efficient genotyping system. The facility currently consists of five liquid handling robots, four
Illumina SherlockTM scanners and other standard molecular biology laboratory equipment,
and is capable of generating 2 million high quality genotype calls per day.
Description of the GoldenGateTM Assay
The Golden GateTM genotyping assay is based on a highly multiplexed, allele-discriminating,
extension reaction. Two allele-specific oligos (ASOs) and a locus-specific oligo (LSO) are used
to query each SNP. The ASOs hybridize 5í to the SNP, and contain a genomic complementarity
region and a universal PCR priming region. The two ASOs differ from each other at their 3í base
position (SNP interrogating position) and their universal PCR priming site. The LSO contains three
elements: a universal PCR priming site located at the 3í end, distinct from the two ASO universal
priming sites; a genomic complementarity region at the 5í end; and a unique address sequence
for hybridization to a complementary sequence on the array.
The three assay oligos are designed, synthesized and pooled at our production facility. We routinely
create pools of 3,456 oligos that we use to interrogate 1,152 SNPs. The pooled assay oligos are
hybridized to genomic DNA in a carefully controlled process. ASOs whose 3í end base pairs with
the SNP position are efficiently extended by a DNA polymerase. ASOs whose 3í end mismatches
the SNP position are not efficiently extended. Thus, extension by DNA polymerase imparts allele
selectivity to the assay. The successfully extended ASOs terminate at the 5í phosphate of their
cognate LSOs. Upon addition of DNA ligase, the successfully extended ASOs are ligated to the
LSOs. The ligation process combines information carried by the allele-specific extension product
(as determined by the DNA polymerase) with information for hybridization to a bead type in the
SentrixTM array matrix (through the unique address sequence in each LSO). The ASO-LSO product
serves as a template for amplification by PCR. Three universal PCR primers are used. Two of the
universal PCR primers are for the ASOs (each fluorescently labeled with a different dye) and the
third universal PCR primer is for the LSO (biotinylated). After thermocycling, the PCR product is
captured on a solid support, and single stranded fluorescently labeled material is eluted and
hybridized to the SentrixTM array matrix. The unique address sequences in the LSOs will hybridize
to complementary sequences on the beads in the SentrixTM array matrix. This last process de-multiplexes
the information about the genotype calls generated in solution by separation on a SentrixTM array matrix.
Illuminaís SentrixTM Array Matrix
Illuminaís SentrixTM array matrix consists of 96 array bundles configured in a microplate-compatible
format. Each array bundle contains nearly 50,000 individual light-conducting fiber strands. The fiber
strands are chemically etched to create a microscopic well at the end of each strand. Most of the wells
are filled, each with a single 3-micron bead. Covalently attached to each bead are several hundred
thousand copies of an oligonucleotide probe. The probes are designed to be highly specific, avoiding
cross-hybridization with each other and with sequences in the human genome. When assembled into
wells, this library of sequences on beads forms a universal array of capture elements that hybridize to
complementary sequences in multiplexed SNP assays. Our current arrays contain 1,520 unique bead
types represented with 30-fold average redundancy in each random array (the actual distribution of bead
types is close to Poisson). Consequently, each SNP genotype determination is the result of data
averaged from multiple beads, greatly reducing the possibility of error.
Since the beads are randomly assembled into arrays, it is necessary to ìdecodeî each array, to identify
the bead at each location, in all 96 arrays in a SentrixTM array matrix. This decoding process consists of
successive hybridization steps with different fluorescently labeled oligonucleotides and results in a
custom bead map file that travels with each SentrixTM array matrix. An important benefit of the decoding
procedure is that each bead is functionally tested before being used in a genotyping assay. This quality
control of every bead in every array bundle is a substantial improvement over batch sampling and provides
a level of quality control without peer in the microarray industry.
SherlockTM scanner
The SentrixTM array matrix is the highest density microarray in commercial use, requiring development of
a high-performance array scanner. To meet this need, we developed the SherlockTM scanner, a laser-scanning
confocal imaging system that automatically scans all 96 bundles of a SentrixTM array matrix at 0.85-micron
resolution. The SherlockTM scans with 532 and 635 nm lasers simultaneously, collecting two fluorescence
images. The SherlockTM scanner software, given the custom bead map file for a SentrixTM array matrix, will
automatically extract intensity information for each bead type in each channel. This information is used along
with SNP assay design information to determine genotypes.
Brief descriptions of each of the steps performed by the Illumina Genotyping Facility are provided below.
Pre-PCR Protocols
1. Genomic DNA preparation
Genomic DNA (gDNA) is activated for binding to a solid support, and is bound to paramagnetic particles. Bound
gDNA is then washed and resuspended in preparation for subsequent steps.
2. Make ASE (make Allele-Specific Extension plate)
A previously prepared oligo pool (containing sets of the three assay oligos) is dispensed into an assay plate along
with hybridization reagents. The resulting ASE plate is ready for the Inoc ASE process. We routinely create and
use oligo pools that allow us to determine genotype calls at 1,152 different SNP positions simultaneously.
3. Inoc ASE (ASE plate inoculation)
Bound gDNAs from the genomic DNA preparation step are added to the oligo pools in the ASE plate. The oligos
are hybridized to the bound gDNA.
4. Add MME (Master Mix for Extension) and MML (Master Mix for Ligation)
A series of washes of the genomic DNA in the ASE plate is performed to remove the excess oligos. A master
mix for extension is then added to extend the allele-specific oligos. After the extension reaction is complete,
the excess extension mix is removed and a master mix for ligation is added so that the extended ASOs may be
ligated to LSOs. The ASE plate is incubated to allow ligation of the extended ASO to the LSO.
5. Make PCR
The Make PCR protocol dispenses PCR master mix from a tube into each well of a 96 well PCR plate. The PCR
master mix contains two fluorescently labeled universal PCR primers and a biotinylated primer. Thermostable DNA
polymerase is added to each tube before the process begins.
6. Inoc PCR (PCR Inoculation)
The extended and ligated products (ASO-LSO product) from the Add MME and MML process in the ASE plate are
transferred to the PCR plate. The Inoc PCR process consists of removing the master mix for ligation from the ASE
plate, followed by the addition of an elution buffer. Heat is used to elute the ASO-LSO product from the bound genomic
DNA, and the eluate containing the ASO-LSO product is transferred to the PCR plate.
Post-PCR Protocols
1. Thermal Cycle PCR
In the Thermal Cycle PCR process, ASO-LSO products are amplified, resulting in double-stranded PCR products
containing a fluor-labeled strand (Cy3 or Cy5, depending on the genotype) and a biotinylated strand (LSO).
2. Bind PCR
The Bind PCR process immobilizes the double-stranded PCR products. Paramagnetic particles are added to the
PCR products and incubated. The biotinylated PCR product is bound to paramagnetic particles.
3. Make HYB
The Make Hyb protocol transfers single-stranded, fluor-labeled PCR product from the PCR plate to a standard HYB
plate suitable for mating to the SentrixTM Array Matrix (SAMs).
4. HYB SentrixTM Array Matrix
The Hyb SentrixTM Array protocol hybridizes single-stranded PCR products to the fiber optic bundles of the SAM.
The HYB plate is mated to a SAM and allowed to incubate. The incubation allows the single-stranded, fluor-labeled
PCR products to hybridize to their complementary sequences on the beads of the SAM.
5. Image SentrixTM Array Matrix
The final step in the SNP genotyping process, Image SentrixTM Array Matrix, washes the hybridized SAM and images
the matrix on Illuminaís SherlockTM 1000 array scanner. The resulting images are stored on a computer network and
analyzed using Illuminaís image analysis software.
LSID: urn:LSID:wicgr.hapmap.org:Protocol:Golden_Gate_1.0.0:1
Title: The Illumina Genotyping Facility
Description:
Shen R, Rubano T, Fan JB, Oliphant A. Optimizing Production-Scale Genotyping. Assay
Tutorial: High-Multiplex SNP Genotyping Assay Benefits from Integration with Turnkey
Production System. Genetic Engineering News 23(6), 2003.
The Illumina Genotyping Facility is based on an informatically integrated production process.
Standard off-the-shelf robotics and laboratory equipment are combined with Illuminaís Golden
GateTM assay, SentrixTM array matrices and SherlockTM scanner technology to form a highly
efficient genotyping system. The facility currently consists of five liquid handling robots, four
Illumina SherlockTM scanners and other standard molecular biology laboratory equipment,
and is capable of generating 2 million high quality genotype calls per day.
Description of the GoldenGateTM Assay
The Golden GateTM genotyping assay is based on a highly multiplexed, allele-discriminating,
extension reaction. Two allele-specific oligos (ASOs) and a locus-specific oligo (LSO) are used
to query each SNP. The ASOs hybridize 5í to the SNP, and contain a genomic complementarity
region and a universal PCR priming region. The two ASOs differ from each other at their 3í base
position (SNP interrogating position) and their universal PCR priming site. The LSO contains three
elements: a universal PCR priming site located at the 3í end, distinct from the two ASO universal
priming sites; a genomic complementarity region at the 5í end; and a unique address sequence
for hybridization to a complementary sequence on the array.
The three assay oligos are designed, synthesized and pooled at our production facility. We routinely
create pools of 3,456 oligos that we use to interrogate 1,152 SNPs. The pooled assay oligos are
hybridized to genomic DNA in a carefully controlled process. ASOs whose 3í end base pairs with
the SNP position are efficiently extended by a DNA polymerase. ASOs whose 3í end mismatches
the SNP position are not efficiently extended. Thus, extension by DNA polymerase imparts allele
selectivity to the assay. The successfully extended ASOs terminate at the 5í phosphate of their
cognate LSOs. Upon addition of DNA ligase, the successfully extended ASOs are ligated to the
LSOs. The ligation process combines information carried by the allele-specific extension product
(as determined by the DNA polymerase) with information for hybridization to a bead type in the
SentrixTM array matrix (through the unique address sequence in each LSO). The ASO-LSO product
serves as a template for amplification by PCR. Three universal PCR primers are used. Two of the
universal PCR primers are for the ASOs (each fluorescently labeled with a different dye) and the
third universal PCR primer is for the LSO (biotinylated). After thermocycling, the PCR product is
captured on a solid support, and single stranded fluorescently labeled material is eluted and
hybridized to the SentrixTM array matrix. The unique address sequences in the LSOs will hybridize
to complementary sequences on the beads in the SentrixTM array matrix. This last process de-multiplexes
the information about the genotype calls generated in solution by separation on a SentrixTM array matrix.
Illuminaís SentrixTM Array Matrix
Illuminaís SentrixTM array matrix consists of 96 array bundles configured in a microplate-compatible
format. Each array bundle contains nearly 50,000 individual light-conducting fiber strands. The fiber
strands are chemically etched to create a microscopic well at the end of each strand. Most of the wells
are filled, each with a single 3-micron bead. Covalently attached to each bead are several hundred
thousand copies of an oligonucleotide probe. The probes are designed to be highly specific, avoiding
cross-hybridization with each other and with sequences in the human genome. When assembled into
wells, this library of sequences on beads forms a universal array of capture elements that hybridize to
complementary sequences in multiplexed SNP assays. Our current arrays contain 1,520 unique bead
types represented with 30-fold average redundancy in each random array (the actual distribution of bead
types is close to Poisson). Consequently, each SNP genotype determination is the result of data
averaged from multiple beads, greatly reducing the possibility of error.
Since the beads are randomly assembled into arrays, it is necessary to ìdecodeî each array, to identify
the bead at each location, in all 96 arrays in a SentrixTM array matrix. This decoding process consists of
successive hybridization steps with different fluorescently labeled oligonucleotides and results in a
custom bead map file that travels with each SentrixTM array matrix. An important benefit of the decoding
procedure is that each bead is functionally tested before being used in a genotyping assay. This quality
control of every bead in every array bundle is a substantial improvement over batch sampling and provides
a level of quality control without peer in the microarray industry.
SherlockTM scanner
The SentrixTM array matrix is the highest density microarray in commercial use, requiring development of
a high-performance array scanner. To meet this need, we developed the SherlockTM scanner, a laser-scanning
confocal imaging system that automatically scans all 96 bundles of a SentrixTM array matrix at 0.85-micron
resolution. The SherlockTM scans with 532 and 635 nm lasers simultaneously, collecting two fluorescence
images. The SherlockTM scanner software, given the custom bead map file for a SentrixTM array matrix, will
automatically extract intensity information for each bead type in each channel. This information is used along
with SNP assay design information to determine genotypes.
Brief descriptions of each of the steps performed by the Illumina Genotyping Facility are provided below.
Pre-PCR Protocols
1. Genomic DNA preparation
Genomic DNA (gDNA) is activated for binding to a solid support, and is bound to paramagnetic particles. Bound
gDNA is then washed and resuspended in preparation for subsequent steps.
2. Make ASE (make Allele-Specific Extension plate)
A previously prepared oligo pool (containing sets of the three assay oligos) is dispensed into an assay plate along
with hybridization reagents. The resulting ASE plate is ready for the Inoc ASE process. We routinely create and
use oligo pools that allow us to determine genotype calls at 1,152 different SNP positions simultaneously.
3. Inoc ASE (ASE plate inoculation)
Bound gDNAs from the genomic DNA preparation step are added to the oligo pools in the ASE plate. The oligos
are hybridized to the bound gDNA.
4. Add MME (Master Mix for Extension) and MML (Master Mix for Ligation)
A series of washes of the genomic DNA in the ASE plate is performed to remove the excess oligos. A master
mix for extension is then added to extend the allele-specific oligos. After the extension reaction is complete,
the excess extension mix is removed and a master mix for ligation is added so that the extended ASOs may be
ligated to LSOs. The ASE plate is incubated to allow ligation of the extended ASO to the LSO.
5. Make PCR
The Make PCR protocol dispenses PCR master mix from a tube into each well of a 96 well PCR plate. The PCR
master mix contains two fluorescently labeled universal PCR primers and a biotinylated primer. Thermostable DNA
polymerase is added to each tube before the process begins.
6. Inoc PCR (PCR Inoculation)
The extended and ligated products (ASO-LSO product) from the Add MME and MML process in the ASE plate are
transferred to the PCR plate. The Inoc PCR process consists of removing the master mix for ligation from the ASE
plate, followed by the addition of an elution buffer. Heat is used to elute the ASO-LSO product from the bound genomic
DNA, and the eluate containing the ASO-LSO product is transferred to the PCR plate.
Post-PCR Protocols
1. Thermal Cycle PCR
In the Thermal Cycle PCR process, ASO-LSO products are amplified, resulting in double-stranded PCR products
containing a fluor-labeled strand (Cy3 or Cy5, depending on the genotype) and a biotinylated strand (LSO).
2. Bind PCR
The Bind PCR process immobilizes the double-stranded PCR products. Paramagnetic particles are added to the
PCR products and incubated. The biotinylated PCR product is bound to paramagnetic particles.
3. Make HYB
The Make Hyb protocol transfers single-stranded, fluor-labeled PCR product from the PCR plate to a standard HYB
plate suitable for mating to the SentrixTM Array Matrix (SAMs).
4. HYB SentrixTM Array Matrix
The Hyb SentrixTM Array protocol hybridizes single-stranded PCR products to the fiber optic bundles of the SAM.
The HYB plate is mated to a SAM and allowed to incubate. The incubation allows the single-stranded, fluor-labeled
PCR products to hybridize to their complementary sequences on the beads of the SAM.
5. Image SentrixTM Array Matrix
The final step in the SNP genotyping process, Image SentrixTM Array Matrix, washes the hybridized SAM and images
the matrix on Illuminaís SherlockTM 1000 array scanner. The resulting images are stored on a computer network and
analyzed using Illuminaís image analysis software.
LSID: urn:LSID:illumina.hapmap.org:Protocol:Golden_Gate_1.1.0:1
Title: The Illumina Genotyping Facility
Description:
The Illumina Genotyping Facility is based on an informatically integrated production process.
Standard off-the-shelf robotics and laboratory equipment are combined with Illumina's Golden
GateTM assay, SentrixTM array matrices and SherlockTM scanner technology to form a highly efficient
genotyping system. The facility currently consists of five liquid handling robots, four Illumina
SherlockTM scanners and other standard molecular biology laboratory equipment, and is capable of
generating 2 million high quality genotype calls per day.
Description of the GoldenGateTM Assay
The Golden GateTM genotyping assay is based on a highly multiplexed, allele-discriminating,
extension reaction. Two allele-specific oligos (ASOs) and a locus-specific oligo (LSO) are
used to query each SNP. The ASOs hybridize 5í to the SNP, and contain a genomic complementarity
region and a universal PCR priming region. The two ASOs differ from each other at their 3í base
position (SNP interrogating position) and their universal PCR priming site. The LSO contains
three elements: a universal PCR priming site located at the 3í end, distinct from the two ASO
universal priming sites; a genomic complementarity region at the 5í end; and a unique address
sequence for hybridization to a complementary sequence on the array.
The three assay oligos are designed, synthesized and pooled at our production facility. We routinely
create pools of 4,608 oligos that we use to interrogate 1,536 SNPs. The pooled assay oligos are
hybridized to genomic DNA in a carefully controlled process. ASOs whose 3í end base pairs with the
SNP position are efficiently extended by a DNA polymerase. ASOs whose 3í end mismatches the SNP position
are not efficiently extended. Thus, extension by DNA polymerase imparts allele selectivity to the assay.
The successfully extended ASOs terminate at the 5í phosphate of their cognate LSOs. Upon addition of DNA
ligase, the successfully extended ASOs are ligated to the LSOs. The ligation process combines information
carried by the allele-specific extension product (as determined by the DNA polymerase) with information
for hybridization to a bead type in the SentrixTM array matrix (through the unique address sequence in
each LSO). The ASO-LSO product serves as a template for amplification by PCR. Three universal PCR primers
are used. Two of the universal PCR primers are for the ASOs (each fluorescently labeled with a different
dye) and the third universal PCR primer is for the LSO (biotinylated). After thermocycling, the PCR product
is captured on a solid support, and single stranded fluorescently labeled material is eluted and hybridized
to the SentrixTM array matrix. The unique address sequences in the LSOs will hybridize to complementary
sequences on the beads in the SentrixTM array matrix. This last process de-multiplexes the information
about the genotype calls generated in solution by separation on a SentrixTM array matrix.
Illuminaís SentrixTM Array Matrix
Illuminaís SentrixTM array matrix consists of 96 array bundles configured in a microplate-compatible
format. Each array bundle contains nearly 50,000 individual light-conducting fiber strands. The fiber
strands are chemically etched to create a microscopic well at the end of each strand. Most of the wells
are filled, each with a single 3-micron bead. Covalently attached to each bead are several hundred thousand
copies of an oligonucleotide probe. The probes are designed to be highly specific, avoiding cross-hybridization
with each other and with sequences in the human genome. When assembled into wells, this library of sequences
on beads forms a universal array of capture elements that hybridize to complementary sequences in multiplexed
SNP assays. Our current arrays contain 1,620 unique bead types represented with 30-fold average redundancy
in each random array (the actual distribution of bead types is close to Poisson). Consequently, each SNP
genotype determination is the result of data averaged from multiple beads, greatly reducing the possibility of
error.
Since the beads are randomly assembled into arrays, it is necessary to ìdecodeî each array, to identify the
bead at each location, in all 96 arrays in a SentrixTM array matrix. This decoding process consists of successive
hybridization steps with different fluorescently labeled oligonucleotides and results in a custom bead map file
that travels with each SentrixTM array matrix. An important benefit of the decoding procedure is that each bead
is functionally tested before being used in a genotyping assay. This quality control of every bead in every array
bundle is a substantial improvement over batch sampling and provides a level of quality control without peer in
the microarray industry.
SherlockTM scanner
The SentrixTM array matrix is the highest density microarray in commercial use, requiring development of a
high-performance array scanner. To meet this need, we developed the SherlockTM scanner, a laser-scanning confocal
imaging system that automatically scans all 96 bundles of a SentrixTM array matrix at 0.85-micron resolution.
The SherlockTM scans with 532 and 635 nm lasers simultaneously, collecting two fluorescence images. The SherlockTM
scanner software, given the custom bead map file for a SentrixTM array matrix, will automatically extract intensity
information for each bead type in each channel. This information is used along with SNP assay design information
to determine genotypes.
Brief descriptions of each of the steps performed by the Illumina Genotyping Facility are provided below.
Pre-PCR Protocols
1. Genomic DNA preparation
Genomic DNA (gDNA) is activated for binding to a solid support.
2. Make ASE (make Allele-Specific Extension plate)
A previously prepared oligo pool (containing sets of the three assay oligos) is dispensed into an assay plate
along with hybridization reagents containing paramagnetic particles and activated gDNA. The activated gDNA is
bound to paramagnetic particles and the oligos hybridized to the bound gDNA.
We routinely create and use oligo pools that allow us to determine genotype calls at 1,536 different SNP positions
simultaneously.
3. Add MEL (Master Mix for Extension and Ligation)
A series of washes of the genomic DNA in the ASE plate are performed to remove the excess oligos. A master mix,
complete for both extension and ligation, is added to extend the allele-specific oligos. The ASE plate is
incubated to allow ligation of the extended ASO to the LSO.
4. Make PCR
The Make PCR protocol dispenses PCR master mix from a tube into each well of a 96 well PCR plate. The PCR master
mix contains two fluorescently labeled universal PCR primers and a biotinylated primer. Thermostable DNA polymerase
is added to each tube before the process begins.
5. Inoc PCR (PCR Inoculation)
The extended and ligated products (ASO-LSO product) from the Add MME and MML process in the ASE plate are transferred
to the PCR plate. The Inoc PCR process consists of removing the master mix for ligation from the ASE plate, followed
by the addition of an elution buffer. Heat is used to elute the ASO-LSO product from the bound genomic DNA, and the
eluate containing the ASO-LSO product is transferred to the PCR plate.
Post-PCR Protocols
1. Thermal Cycle PCR
In the Thermal Cycle PCR process, ASO-LSO products are amplified, resulting in double-stranded PCR products containing
a fluor-labeled strand (Cy3 or Cy5, depending on the genotype) and a biotinylated strand (LSO).
2. Bind PCR
The Bind PCR process immobilizes the double-stranded PCR products. Paramagnetic particles are added to the PCR products
and incubated. The biotinylated PCR product is bound to paramagnetic particles.
3. Make HYB
The Make Hyb protocol transfers single-stranded, fluor-labeled PCR product from the PCR plate to a standard HYB plate
suitable for mating to the SentrixTM Array Matrix (SAMs).
4. HYB SentrixTM Array Matrix
The Hyb SentrixTM Array protocol hybridizes single-stranded PCR products to the fiber optic bundles of the SAM. The
HYB plate is mated to a SAM and allowed to incubate. The incubation allows the single-stranded, fluor-labeled PCR
products to hybridize to their complementary sequences on the beads of the SAM.
5. Image SentrixTM Array Matrix
The final step in the SNP genotyping process, Image SentrixTM Array Matrix, washes the hybridized SAM and images the
matrix on Illuminaís SherlockTM 1000 array scanner. The resulting images are stored on a computer network and analyzed
using Illuminaís image analysis software.
LSID: urn:LSID:mcgill-gqic.hapmap.org:Protocol:Golden_Gate_1.0.0:1
Title: The Illumina Genotyping Facility
Description:
Shen R, Rubano T, Fan JB, Oliphant A. Optimizing Production-Scale Genotyping. Assay
Tutorial: High-Multiplex SNP Genotyping Assay Benefits from Integration with Turnkey
Production System. Genetic Engineering News 23(6), 2003.
The Illumina Genotyping Facility is based on an informatically integrated production process.
Standard off-the-shelf robotics and laboratory equipment are combined with Illuminaís Golden
GateTM assay, SentrixTM array matrices and SherlockTM scanner technology to form a highly
efficient genotyping system. The facility currently consists of five liquid handling robots, four
Illumina SherlockTM scanners and other standard molecular biology laboratory equipment,
and is capable of generating 2 million high quality genotype calls per day.
Description of the GoldenGateTM Assay
The Golden GateTM genotyping assay is based on a highly multiplexed, allele-discriminating,
extension reaction. Two allele-specific oligos (ASOs) and a locus-specific oligo (LSO) are used
to query each SNP. The ASOs hybridize 5í to the SNP, and contain a genomic complementarity
region and a universal PCR priming region. The two ASOs differ from each other at their 3í base
position (SNP interrogating position) and their universal PCR priming site. The LSO contains three
elements: a universal PCR priming site located at the 3í end, distinct from the two ASO universal
priming sites; a genomic complementarity region at the 5í end; and a unique address sequence
for hybridization to a complementary sequence on the array.
The three assay oligos are designed, synthesized and pooled at our production facility. We routinely
create pools of 3,456 oligos that we use to interrogate 1,152 SNPs. The pooled assay oligos are
hybridized to genomic DNA in a carefully controlled process. ASOs whose 3í end base pairs with
the SNP position are efficiently extended by a DNA polymerase. ASOs whose 3í end mismatches
the SNP position are not efficiently extended. Thus, extension by DNA polymerase imparts allele
selectivity to the assay. The successfully extended ASOs terminate at the 5í phosphate of their
cognate LSOs. Upon addition of DNA ligase, the successfully extended ASOs are ligated to the
LSOs. The ligation process combines information carried by the allele-specific extension product
(as determined by the DNA polymerase) with information for hybridization to a bead type in the
SentrixTM array matrix (through the unique address sequence in each LSO). The ASO-LSO product
serves as a template for amplification by PCR. Three universal PCR primers are used. Two of the
universal PCR primers are for the ASOs (each fluorescently labeled with a different dye) and the
third universal PCR primer is for the LSO (biotinylated). After thermocycling, the PCR product is
captured on a solid support, and single stranded fluorescently labeled material is eluted and
hybridized to the SentrixTM array matrix. The unique address sequences in the LSOs will hybridize
to complementary sequences on the beads in the SentrixTM array matrix. This last process de-multiplexes
the information about the genotype calls generated in solution by separation on a SentrixTM array matrix.
Illuminaís SentrixTM Array Matrix
Illuminaís SentrixTM array matrix consists of 96 array bundles configured in a microplate-compatible
format. Each array bundle contains nearly 50,000 individual light-conducting fiber strands. The fiber
strands are chemically etched to create a microscopic well at the end of each strand. Most of the wells
are filled, each with a single 3-micron bead. Covalently attached to each bead are several hundred
thousand copies of an oligonucleotide probe. The probes are designed to be highly specific, avoiding
cross-hybridization with each other and with sequences in the human genome. When assembled into
wells, this library of sequences on beads forms a universal array of capture elements that hybridize to
complementary sequences in multiplexed SNP assays. Our current arrays contain 1,520 unique bead
types represented with 30-fold average redundancy in each random array (the actual distribution of bead
types is close to Poisson). Consequently, each SNP genotype determination is the result of data
averaged from multiple beads, greatly reducing the possibility of error.
Since the beads are randomly assembled into arrays, it is necessary to ìdecodeî each array, to identify
the bead at each location, in all 96 arrays in a SentrixTM array matrix. This decoding process consists of
successive hybridization steps with different fluorescently labeled oligonucleotides and results in a
custom bead map file that travels with each SentrixTM array matrix. An important benefit of the decoding
procedure is that each bead is functionally tested before being used in a genotyping assay. This quality
control of every bead in every array bundle is a substantial improvement over batch sampling and provides
a level of quality control without peer in the microarray industry.
SherlockTM scanner
The SentrixTM array matrix is the highest density microarray in commercial use, requiring development of
a high-performance array scanner. To meet this need, we developed the SherlockTM scanner, a laser-scanning
confocal imaging system that automatically scans all 96 bundles of a SentrixTM array matrix at 0.85-micron
resolution. The SherlockTM scans with 532 and 635 nm lasers simultaneously, collecting two fluorescence
images. The SherlockTM scanner software, given the custom bead map file for a SentrixTM array matrix, will
automatically extract intensity information for each bead type in each channel. This information is used along
with SNP assay design information to determine genotypes.
Brief descriptions of each of the steps performed by the Illumina Genotyping Facility are provided below.
Pre-PCR Protocols
1. Genomic DNA preparation
Genomic DNA (gDNA) is activated for binding to a solid support, and is bound to paramagnetic particles. Bound
gDNA is then washed and resuspended in preparation for subsequent steps.
2. Make ASE (make Allele-Specific Extension plate)
A previously prepared oligo pool (containing sets of the three assay oligos) is dispensed into an assay plate along
with hybridization reagents. The resulting ASE plate is ready for the Inoc ASE process. We routinely create and
use oligo pools that allow us to determine genotype calls at 1,152 different SNP positions simultaneously.
3. Inoc ASE (ASE plate inoculation)
Bound gDNAs from the genomic DNA preparation step are added to the oligo pools in the ASE plate. The oligos
are hybridized to the bound gDNA.
4. Add MME (Master Mix for Extension) and MML (Master Mix for Ligation)
A series of washes of the genomic DNA in the ASE plate is performed to remove the excess oligos. A master
mix for extension is then added to extend the allele-specific oligos. After the extension reaction is complete,
the excess extension mix is removed and a master mix for ligation is added so that the extended ASOs may be
ligated to LSOs. The ASE plate is incubated to allow ligation of the extended ASO to the LSO.
5. Make PCR
The Make PCR protocol dispenses PCR master mix from a tube into each well of a 96 well PCR plate. The PCR
master mix contains two fluorescently labeled universal PCR primers and a biotinylated primer. Thermostable DNA
polymerase is added to each tube before the process begins.
6. Inoc PCR (PCR Inoculation)
The extended and ligated products (ASO-LSO product) from the Add MME and MML process in the ASE plate are
transferred to the PCR plate. The Inoc PCR process consists of removing the master mix for ligation from the ASE
plate, followed by the addition of an elution buffer. Heat is used to elute the ASO-LSO product from the bound genomic
DNA, and the eluate containing the ASO-LSO product is transferred to the PCR plate.
Post-PCR Protocols
1. Thermal Cycle PCR
In the Thermal Cycle PCR process, ASO-LSO products are amplified, resulting in double-stranded PCR products
containing a fluor-labeled strand (Cy3 or Cy5, depending on the genotype) and a biotinylated strand (LSO).
2. Bind PCR
The Bind PCR process immobilizes the double-stranded PCR products. Paramagnetic particles are added to the
PCR products and incubated. The biotinylated PCR product is bound to paramagnetic particles.
3. Make HYB
The Make Hyb protocol transfers single-stranded, fluor-labeled PCR product from the PCR plate to a standard HYB
plate suitable for mating to the SentrixTM Array Matrix (SAMs).
4. HYB SentrixTM Array Matrix
The Hyb SentrixTM Array protocol hybridizes single-stranded PCR products to the fiber optic bundles of the SAM.
The HYB plate is mated to a SAM and allowed to incubate. The incubation allows the single-stranded, fluor-labeled
PCR products to hybridize to their complementary sequences on the beads of the SAM.
5. Image SentrixTM Array Matrix
The final step in the SNP genotyping process, Image SentrixTM Array Matrix, washes the hybridized SAM and images
the matrix on Illuminaís SherlockTM 1000 array scanner. The resulting images are stored on a computer network and
analyzed using Illuminaís image analysis software.
LSID: urn:LSID:illumina.hapmap.org:Protocol:Golden_Gate_genotyping_1.0.0:1
Title: The Illumina Genotyping Facility
Description:
Shen R, Rubano T, Fan JB, Oliphant A. ìOptimizing Production-Scale Genotyping. Assay
Tutorial: High-Multiplex SNP Genotyping Assay Benefits from Integration with Turnkey
Production System. Genetic Engineering News 23(6), 2003.
The Illumina Genotyping Facility is based on an informatically integrated production process.
Standard off-the-shelf robotics and laboratory equipment are combined with Illuminaís Golden
GateTM assay, SentrixTM array matrices and SherlockTM scanner technology to form a highly
efficient genotyping system. The facility currently consists of five liquid handling robots, four
Illumina SherlockTM scanners and other standard molecular biology laboratory equipment,
and is capable of generating 2 million high quality genotype calls per day.
Description of the GoldenGateTM Assay
The Golden GateTM genotyping assay is based on a highly multiplexed, allele-discriminating,
extension reaction. Two allele-specific oligos (ASOs) and a locus-specific oligo (LSO) are used
to query each SNP. The ASOs hybridize 5í to the SNP, and contain a genomic complementarity
region and a universal PCR priming region. The two ASOs differ from each other at their 3í base
position (SNP interrogating position) and their universal PCR priming site. The LSO contains three
elements: a universal PCR priming site located at the 3í end, distinct from the two ASO universal
priming sites; a genomic complementarity region at the 5í end; and a unique address sequence
for hybridization to a complementary sequence on the array.
The three assay oligos are designed, synthesized and pooled at our production facility. We routinely
create pools of 3,456 oligos that we use to interrogate 1,152 SNPs. The pooled assay oligos are
hybridized to genomic DNA in a carefully controlled process. ASOs whose 3í end base pairs with
the SNP position are efficiently extended by a DNA polymerase. ASOs whose 3í end mismatches
the SNP position are not efficiently extended. Thus, extension by DNA polymerase imparts allele
selectivity to the assay. The successfully extended ASOs terminate at the 5í phosphate of their
cognate LSOs. Upon addition of DNA ligase, the successfully extended ASOs are ligated to the
LSOs. The ligation process combines information carried by the allele-specific extension product
(as determined by the DNA polymerase) with information for hybridization to a bead type in the
SentrixTM array matrix (through the unique address sequence in each LSO). The ASO-LSO product
serves as a template for amplification by PCR. Three universal PCR primers are used. Two of the
universal PCR primers are for the ASOs (each fluorescently labeled with a different dye) and the
third universal PCR primer is for the LSO (biotinylated). After thermocycling, the PCR product is
captured on a solid support, and single stranded fluorescently labeled material is eluted and
hybridized to the SentrixTM array matrix. The unique address sequences in the LSOs will hybridize
to complementary sequences on the beads in the SentrixTM array matrix. This last process de-multiplexes
the information about the genotype calls generated in solution by separation on a SentrixTM array matrix.
Illuminaís SentrixTM Array Matrix
Illuminaís SentrixTM array matrix consists of 96 array bundles configured in a microplate-compatible
format. Each array bundle contains nearly 50,000 individual light-conducting fiber strands. The fiber
strands are chemically etched to create a microscopic well at the end of each strand. Most of the wells
are filled, each with a single 3-micron bead. Covalently attached to each bead are several hundred
thousand copies of an oligonucleotide probe. The probes are designed to be highly specific, avoiding
cross-hybridization with each other and with sequences in the human genome. When assembled into
wells, this library of sequences on beads forms a universal array of capture elements that hybridize to
complementary sequences in multiplexed SNP assays. Our current arrays contain 1,520 unique bead
types represented with 30-fold average redundancy in each random array (the actual distribution of bead
types is close to Poisson). Consequently, each SNP genotype determination is the result of data
averaged from multiple beads, greatly reducing the possibility of error.
Since the beads are randomly assembled into arrays, it is necessary to ìdecodeî each array, to identify
the bead at each location, in all 96 arrays in a SentrixTM array matrix. This decoding process consists of
successive hybridization steps with different fluorescently labeled oligonucleotides and results in a
custom bead map file that travels with each SentrixTM array matrix. An important benefit of the decoding
procedure is that each bead is functionally tested before being used in a genotyping assay. This quality
control of every bead in every array bundle is a substantial improvement over batch sampling and provides
a level of quality control without peer in the microarray industry.
SherlockTM scanner
The SentrixTM array matrix is the highest density microarray in commercial use, requiring development of
a high-performance array scanner. To meet this need, we developed the SherlockTM scanner, a laser-scanning
confocal imaging system that automatically scans all 96 bundles of a SentrixTM array matrix at 0.85-micron
resolution. The SherlockTM scans with 532 and 635 nm lasers simultaneously, collecting two fluorescence
images. The SherlockTM scanner software, given the custom bead map file for a SentrixTM array matrix, will
automatically extract intensity information for each bead type in each channel. This information is used along
with SNP assay design information to determine genotypes.
Brief descriptions of each of the steps performed by the Illumina Genotyping Facility are provided below.
Pre-PCR Protocols
1. Genomic DNA preparation
Genomic DNA (gDNA) is activated for binding to a solid support, and is bound to paramagnetic particles. Bound
gDNA is then washed and resuspended in preparation for subsequent steps.
2. Make ASE (make Allele-Specific Extension plate)
A previously prepared oligo pool (containing sets of the three assay oligos) is dispensed into an assay plate along
with hybridization reagents. The resulting ASE plate is ready for the Inoc ASE process. We routinely create and
use oligo pools that allow us to determine genotype calls at 1,152 different SNP positions simultaneously.
3. Inoc ASE (ASE plate inoculation)
Bound gDNAs from the genomic DNA preparation step are added to the oligo pools in the ASE plate. The oligos
are hybridized to the bound gDNA.
4. Add MME (Master Mix for Extension) and MML (Master Mix for Ligation)
A series of washes of the genomic DNA in the ASE plate is performed to remove the excess oligos. A master
mix for extension is then added to extend the allele-specific oligos. After the extension reaction is complete,
the excess extension mix is removed and a master mix for ligation is added so that the extended ASOs may be
ligated to LSOs. The ASE plate is incubated to allow ligation of the extended ASO to the LSO.
5. Make PCR
The Make PCR protocol dispenses PCR master mix from a tube into each well of a 96 well PCR plate. The PCR
master mix contains two fluorescently labeled universal PCR primers and a biotinylated primer. Thermostable DNA
polymerase is added to each tube before the process begins.
6. Inoc PCR (PCR Inoculation)
The extended and ligated products (ASO-LSO product) from the Add MME and MML process in the ASE plate are
transferred to the PCR plate. The Inoc PCR process consists of removing the master mix for ligation from the ASE
plate, followed by the addition of an elution buffer. Heat is used to elute the ASO-LSO product from the bound genomic
DNA, and the eluate containing the ASO-LSO product is transferred to the PCR plate.
Post-PCR Protocols
1. Thermal Cycle PCR
In the Thermal Cycle PCR process, ASO-LSO products are amplified, resulting in double-stranded PCR products
containing a fluor-labeled strand (Cy3 or Cy5, depending on the genotype) and a biotinylated strand (LSO).
2. Bind PCR
The Bind PCR process immobilizes the double-stranded PCR products. Paramagnetic particles are added to the
PCR products and incubated. The biotinylated PCR product is bound to paramagnetic particles.
3. Make HYB
The Make Hyb protocol transfers single-stranded, fluor-labeled PCR product from the PCR plate to a standard HYB
plate suitable for mating to the SentrixTM Array Matrix (SAMs).
4. HYB SentrixTM Array Matrix
The Hyb SentrixTM Array protocol hybridizes single-stranded PCR products to the fiber optic bundles of the SAM.
The HYB plate is mated to a SAM and allowed to incubate. The incubation allows the single-stranded, fluor-labeled
PCR products to hybridize to their complementary sequences on the beads of the SAM.
5. Image SentrixTM Array Matrix
The final step in the SNP genotyping process, Image SentrixTM Array Matrix, washes the hybridized SAM and images
the matrix on Illuminaís SherlockTM 1000 array scanner. The resulting images are stored on a computer network and
analyzed using Illuminaís image analysis software.
FP-TDI platform (used by UCSF/WashU)
Defined protocols:
LSID: urn:LSID:ucsf-wu.hapmap.org:Protocol:genotyping:1
Title: FP-TDI Reaction Protocol
Description: Template-directed Dye-terminator Incorporation Assay with Fluorescence Polarization detection
(with dried DNA and EP3 liquid handling)
1. Primer Dilution
Primers come as 6 nmol dried pellets in 96-well plate, add 150ul water to each well to make
40uM stock solution, seal with foil cover, vortex and spin the plates. One set of primers p1, p2 and p3
or p4 are in the same positions of 3 separate plates.
To cover the bottom of the resevoirs 6ml is needed. For PCR primer preparation, take 30 ul of each p1
and p2 add to 5940ul water to make 0.2 uM each working solution (p1 and p2 are mixed).
For SNP primer (p3 or p4), take 150 ul primer and add to 5850 ul water to make 1 uM working solution.
Primers could be dilute into 10 ml tubes or to the reservoirs directly, mix well before use.
Discard PCR primers but recover SNP primers and keep in 4ºC for any possible repeating within 1 week.
"water" is autoclaved distilled water.
All primers should be stored in -20º C when not in use.
2. Make Dried DNA Plates
Dispense 3 ul (0.8ng/ul) DNA from 96-well DNA plate to 4 quadrants of 384 black plates with EP3.
Spin plates and air dry at room tempreture overnight, stack and wrap plates with plastic film and keep
in vacuum desicator for use.
3. PCR
Mix for 12 plates with dead volume
Mix for exact 12 plates each
X4800 X7000
Genomic DNA (0.8ng/ul) dried
10X PCR buffer 0.5 2400 3500
MgCl2 (25mM) 0.5 2400 3500
dNTPs (2.5mM) 0.1 480 700
Taq(2.5U/ul) 0.04 192 280
Water 1.86 8928 13020
6 (ul) 14400(ul) 21000(ul)
a. Dispense 3ul 0.2uM ea. PCR primers to relevant quadrants in 384 plates containing 2.4ng DNA, spin.
b. Dispense 3ul mix to each plate, spin and cover with Microseal 'A' film.
c. Final volume is 6ul, 1ul extra water is added to compensate for evaporation within wells during cycling.
Cycling program
PLT-58
1 95oC 2 min.
2 92 10 sec.
3 58 20 sec.
4 68 30 sec.
5 34 times to 2
6 68 10 min.
7 4 For ever
4. Exo-Sap Reaction
Dilute PCR clean up reagent (10X) 10 times with PCR clean up buffer--both provided by
AcycloPrimer-FP SNP Detection kit. Add 2 ul to each well with EP3, spin.
In order to get better dispensing results, there is no tip washing during Exo-Sap dispensing.
Cycling program
SAPNEW1
1 37ºC 60min
2 80 15min
3 4 forever
5. TDI Reaction
Mix for 12 plates with dead volume
Mix for exact 12 plates
each X4800 X5500
10X buffer 2 9600 11000
AcycloPol 0.05 240 275
Terminator mix 1 4800 5500
Water 4.95 23760 27225
total 8(ul) 38,400(ul) 44,000(ul)
a. Dispense 5 ul of 1uM SNP primers to each relevant quadrants in 384 plates, spin.
b. Dispense 8 ul of TDI mix to the plates, spin.
c. If using different terminator mix in one plate, dispensing program has to be changed to dispense specific quadrants.
d. 10 X buffer, terminator mix and AcycloPol are provided by AcycloPrimer-FP SNP Detection kit.
Cycling program
TDI20NEW
1 95oC 2 min.
2 95 15 sec.
3 55 30 sec.
4 19 times to 2
5 4 For ever
5. Read Plates
Spin the plates, put the barcode on back side of each plate, stack the plates in the Victor stacker.
If not reading immediately, plates could be well sealed and stored at 4º C in the dark.
LSID: urn:lsid:ucsf-wu.hapmap.org:Protocol:genotyping-wu-singleplex-full:1
Title: Genotyping Assay Design for Single Base Extention and FP-TDI Detection
Description: Full-reaction single-plex at WU lab
LSID: urn:lsid:ucsf-wu.hapmap.org:Protocol:genotyping-wu-singleplex-half:1
Title: Genotyping Assay Design for Single Base Extention and FP-TDI Detection
Description: Half-reaction single-plex at WU lab
LSID: urn:lsid:ucsf-wu.hapmap.org:Protocol:genotyping-ucsf-singleplex:1
Title: Genotyping Assay Design for Single Base Extention and FP-TDI Detection
Description: Single-plex at UCSF lab
LSID: urn:lsid:ucsf-wu.hapmap.org:Protocol:genotyping-ucsf-multiplex:1
Title: Genotyping Assay Design for Single Base Extention and FP-TDI Detection
Description: Multi-plex at UCSF lab
Invader platform (used by RIKEN)
Defined protocols:
LSID: urn:LSID:imsut-riken.hapmap.org:Protocol:genotyping:1
Title: Invader Reaction Protocol
Description:
SNPs in each set was simultaneously amplified using 1) Multiplex PCR (96plex) reaction.
Thereafter, 2) Invader Reaction were applied with each SNP, under condition described in
3) Incubation for Invader reaction and 4) Emission Wavelength.
1) Multiplex PCR(96plex)
96 SNPs were selected for one multiplex PCR experiment so that their PCR product length
was almost the same with each other. SNPs in each set was simultaneously amplified using
reagents:
Genomic DNA(10ng/ul) 1.0ul
5x PCR Buffer 2.0ul
Forward Primer Mixture(96 primers, 1pmol/ul each) 2.5ul
Reverse Primer Mixture(96 primers, 1pmol/ul each) 2.5ul
ExTaq(5U/ul) with AntiTaqAntibody(15U/ul) 0.5ul
D.W. 1.5ul
(Total 10.0ul)
with cycling conditions:
94 degrees C 2min
94 degrees C 15sec start of each cycle
60 degrees C 45sec 37cycles
72 degrees C 3min end of each cycle
72 degrees C 7min
10 degrees C
2) Invader Reaction
PCR Product(1/10dilute) 1.0ul(dry up in tube)
10x FRET Probe 0.1ul
10x Cleavase 0.1ul
10x Invader Buffer 0.1ul
5x (Allele Probe + Invader Probe) 0.2ul
0.5mM ROX 0.006ul
D.W. 0.494ul
(Total 1.0ul/assay)
3) Incubation for Invader reaction
96 degrees C 5min
63 degrees C 30min
4) Emission Wavelength
FAM(520nm), VIC(545nm), ROX(610nm)
MIP platform (used by Baylor)
Defined protocols:
LSID: urn:LSID:bcm.hapmap.org:Protocol:genotype_0002:1
Title: MIP Reaction Protocol
Description:
Probe annealing
Prepare for each sample:
Genomic DNA 2 ug
Multiplex probe pool 5ul
Annealing mix 5.0625ul
H2O to 45 ul final volume
Anneal overnight 60 degrees C.
Gap fill
Incubate 100ul of GapFill mix at 20 degrees C for 5 min, 95 degrees C for 5 min, and then equilibrate to 60 degrees C.
Add to each annealed probe 5 ul of equilibrated gap fill mix.
Split reaction into four 10 ul aliquots.
Incubate at 60 degrees C for 10 min.
Add 1ul each of dATP, dCTP, dGTP, dTTP to each of four reactions respectively.
Incubate for 10min at 60 degrees C.
Add 2 ul exonuclease mix
Incubate 15 min at 37 degrees C
Incubate 2 min at 95 degrees C
Equilibrate to 37 degrees C
Ung treatment
Add 25 ul of UNG mix to each reaction (4 reactions per sample)
Incubate 10 min at 37 degrees
Incubate 10 min at 95 degrees
Amplification
Equilibrate 1500ul amplification mix at 95 degrees C for 10 min prior to addition
Add 25 ul amplification mix to each reaction (4 reactions per sample) while both the amp mix and the reaction are at 95 degrees C.
Amplify 26 cycles using the profile:
Degrees Seconds
95 20
64 45
72 10
Hold at 72 degrees for an additional 10 seconds then at 4 degrees indefinitely.
Second amplification
Two mixes are used; one for A and C reactions, one for G and T reactions as below.
1. Amplification mix (A/C) 2970ul
2. Amplification buffer mix (G/T) 2970ul
Add 99ul of amplification mix to 1ul of each respective reaction, totaling 100ul per reaction.
Incubate 10 min at 95 degrees C
Amplify 7 cycles using the profile:
Degrees Seconds
95 20
64 45
72 10
Hold at 72 degrees for an additional 3 min then at 4 degrees indefinitely.
Exo-Dra treatment
Add 6.67ul of Exo-Dra Mix to each reaction.
Incubate 1 hour at 37 degrees C
Incubate 30 min at 80 degrees C
Hold at 4 degrees indefinitely
Consolidation
Per sample, consolidate from 4 reactions to 2 reactions as below.
"A" reaction = 90ul + "G" reaction = 18ul
"C" reaction = 90ul + "T" reaction = 18ul
Add 1ml of unsaturated butanol to each consolidated reaction, mix, centrifuge.
Remove 620ul of saturated butanol from reaction.
Add 620ul of unsaturated butanol to reaction, mix, centrifuge.
Transfer 200ul of concentrated sample and remove any residual saturated butanol.
Hyb
Add 115ul of Hyb Mix to concentrated reaction to a final volume of 165ul.
Incubate 6 min at 95 degrees C
Set on ice for 4 min
Load on Tag3 chips.
Incubate overnight at 39 degrees C in oven rotating at 20 rpm.
Chip Wash and Stain
Remove samples from chips.
Wash as per Affymetrix GeneChip protocol.
Add 135ul stain mix per chip.
Rotate chips in oven with no heat at 25 rpm for 10 min.
Remove stain.
Wash as per Affymetrix GeneChip protocol.
Perlegen platform (used by Perlegen)
Defined protocols:
LSID: urn:lsid:perlegen.hapmap.org:Protocol:Genotyping_1.0.0:1
Title: Perlegen Genotyping Protocol
Description: Light-directed chemical synthesis of oligonucleotide arrays was
carried out by Affymetrix, Inc. (Santa Clara, CA). Genomic DNA was
amplified by multiplex long-range PCR, then all PCR products destined
for a single genotyping array were pooled together and end-labeled
with biotinylated nucleotides. Each labeled DNA sample was hybridized
to a chip in hybridization buffer overnight at 50C. The hybridized
chips were washed several times in neutral buffer at RT, then
incubated with streptavidin for 15 min at RT. Following two washes at
35C in neutral buffer, chips were incubated with biotinylated
anti-streptavidin antibody for 15 min at RT, followed by another two
washes at 35C in neutral buffer. Then, chips were stained with
streptavidin-Cy-chrome conjugate for 15 min at RT, followed by two
washes with neutral buffer at 35C. Chips were incubated 30 min at 37C
in low-salt buffer, followed by a wash with neutral buffer at RT.
Hybridization of the sample to the array was detected using a confocal
laser scanner.
Individual genotypes for a SNP were determined by clustering
measurements from multiple scans in the two-dimensional space defined
by background-adjusted intensities of the perfect-match features for
the reference and alternate alleles. We used a K-means algorithm to
assign measurements to clusters representing distinct diploid
genotypes. Instead of estimating the background intensity from a
single scan, we determined an optimal value for each SNP that
minimized the variance within the assigned genotype clusters. The
K-means and background optimization steps were iterated until cluster
membership and background estimates converged. To determine the
appropriate number of genotype clusters, we repeated the analysis for
1, 2, and 3 clusters, and selected the most likely solution,
considering likelihoods of the data and the cluster parameters. The
data likelihood was determined using a normal mixture model for the
distribution of intensities around the cluster means. The model
likelihood was calculated using a prior distribution of expected
cluster positions.
LSID: urn:lsid:perlegen.hapmap.org:Protocol:Genotyping_1.0.0:2
Title: Perlegen Genotyping Protocol
Description: Light-directed chemical synthesis of oligonucleotide arrays was
carried out by Affymetrix, Inc. (Santa Clara, CA). Genomic DNA was
amplified by multiplex long-range PCR, then all PCR products destined
for a single genotyping array were pooled together and end-labeled
with biotinylated nucleotides. Each labeled DNA sample was hybridized
to a chip in hybridization buffer overnight at 50C. The hybridized
chips were washed several times in neutral buffer at RT, then
incubated with streptavidin for 15 min at RT. Following two washes at
35C in neutral buffer, chips were incubated with biotinylated
anti-streptavidin antibody for 15 min at RT, followed by another two
washes at 35C in neutral buffer. Then, chips were stained with
streptavidin-Cy-chrome conjugate for 15 min at RT, followed by two
washes with neutral buffer at 35C. Chips were incubated 30 min at 37C
in low-salt buffer, followed by a wash with neutral buffer at RT.
Hybridization of the sample to the array was detected using a confocal
laser scanner.
Individual genotypes for a SNP were determined by clustering
measurements from multiple scans in the two-dimensional space defined
by background-adjusted intensities of the perfect-match features for
the reference and alternate alleles. We used a K-means algorithm to
assign measurements to clusters representing distinct diploid
genotypes. Instead of estimating the background intensity from a
single scan, we determined an optimal value for each SNP that
minimized the variance within the assigned genotype clusters. The
K-means and background optimization steps were iterated until cluster
membership and background estimates converged. To determine the
appropriate number of genotype clusters, we repeated the analysis for
1, 2, and 3 clusters, and selected the most likely solution,
considering likelihoods of the data and the cluster parameters. The
data likelihood was determined using a normal mixture model for the
distribution of intensities around the cluster means. The model
likelihood was calculated using a prior distribution of expected
cluster positions.
[same as urn:lsid:perlegen.hapmap.org:Protocol:Genotyping_1.0.0:1 except no duplicated samples on CEU and YRI, and new sample on JPT]
Sequenom platform (used by Broad, Beijing, HongKong)
Defined protocols:
LSID: urn:lsid:wicgr.hapmap.org:Protocol:genotype_protocol_1:1
Title: Genotyping using primer mass extension and MALDI-TOF MS analysis
Description: PCR protocol:
Multiplex PCR reactions were performed using standard conditions in 384 well PCR plates (Eppendorf).
The 6 uL PCR reactionscontain 0.2 U of Taq polymerase (HotStar, Qiagen); 5 ng genomic DNA; 100 nM
(0.6 pmol) of each PCR primer; and 200 uM (1.2 nmol) of each dNTP. Cycling conditions were initial
denaturation at 95oC 15 minutes followed by 45 cycles of 95oC for 20 s, 56oC for 30s, 72oC for 30 s.
Dephosphorylation:
To remove residual unincorporated dNTPs from the PCR reaction, 2 uL mix of 0.3 U of Shrimp Alkaline
Phosphatase (Amersham) and 5X ThermoSequenase buffer (250 mM Tris, 10 mM MgCl2, pH 9.5) was added to
the 6uL PCR reaction, followed by a 25 min incubation at 37oC and heat-inactivation of the enzyme at
85oC for 10 min.
Primer Extension Protocol:
During the MassEXTEND reaction, a probe is extended by a specific number of nucleotides depending
on the allele and the design of the assay. In the reaction mixture, all four nucleotides A, T, C,
and G are present as either dNTPs or ddNTPs. Three nucleotides are present as ddNTPS and one as a
dNTP. The incorporation of a ddNTP terminates the extension of the primer. For each type of SNP an
optimal termination mix is used (C/T SNP uses A, C, G ddNTPs; C/G, A/G, and G/T SNPs use A, C, T
ddNTPs; A/C use C, G, T ddNPTs; A/T uses C, G, T ddNTPs). Using a DNA polymerase that incorporates
both dNTPs and ddNTPs, the MassEXTEND reaction produces alelle-specific extension products of
different masses depending on the sequence analyzed. The reaction consists of 52 uM (5.2 nmol) of
the specific termination mix, 650 nM (~ 6 pmol) of each primer extension probe and 0.64 U of
ThermoSequenase polymerase (Amersham Phamacia). 2 uL of the above reaction mixture was added
directly to the dephosphorylated PCR products bringing the final reaction volume to 10 uL. Cycling
conditions were 94 oC for 2 minutes, followed by 54 cycles of 94oC for 5 s, 50oC for 5 s, 72oC for 5 s.
MALDI-TOF MS analysis:
Approximately 7nl of the purified primer extension reaction was loaded onto a matrix pad (3-
hydroxypicoloinicacid) of a SpectroCHIP (Sequenom, San Diego, CA). SpectroCHIPs were analyzed using
a Bruker Biflex III MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass
spectrometer (SpectroREADER, Sequenom, San Diego, CA). The resulting mass spectra were processed by
proprietary software (SpectroTYPER) to determine genotypes based on peaks intensities corresponding
to the expected extension products.
Generated 10:13:45 13-Oct-2005
Last updated : protocol.tt2,v 1.2 2004/11/25 16:32:25 mummi Exp
