CAPÍTULO 5. RESULTADOS OBTENIDOS
5.6 Idoneidad del uso de un wiki en diversos ámbitos
10µl of GelRed nucleic acid stain gel (VWR cat # 89139-‐140) was added to the heated agarose, and the entire solution was poured into an electrophoresis tank and left to cool for 10 minutes. DNA samples mixed with 5µl loading dye were loaded onto the set gel along with 5µl of HyperLadder 100bp (Bioline cat # 33056); the gel was run at 100V for up to 20 minutes.
Table 2.1: Standard PCR reagents, concentrations and volumes for genomic
DNA
Reagents
(Supplier) Concentration for one reaction Volume for one reaction
Primers
(Sigma Aldrich, 100µM) 10uM 2µl forward and reverse AmpliTaq Gold® PCR Master Mix
includes:
AmpliTaq Gold® DNA polymerase Gold Buffer
MgCl2
(Life Sciences 1000 units cat # 4338858) 5U/µl 10 x 25mM 0.2µl 2µl 2µl dNTPS (Bioline 100mM cat # 39025) 25mM 2µl
Stock genomic DNA 25ng 5µl
Molecular Grade Water
(Sigma Aldrich cat # W4502) -‐ 6.8µl
2.5 Exome target capture
Two target capture methods are outlined in this section: the first method describes array capture with 12-‐sample pooling and multiplex sequencing (Phase One, intended as a pilot study); the second describes single sample capture with exome probes in-‐solution (Phase Two). The same library preparation reagents were used for both methods.
2.5.1 NimbleGen human exome 2.1M array (Phase one)
A pilot experiment was carried out with 60 samples on NimbleGen Sequence Capture Human Exome 2.1M Array, v1 (NimbleGen, USA), containing 180,000 coding exons (CCDS transcripts) and 551 miRNA exons totaling 26.7Mb of the human exome. Samples were multiplexed (12 samples per pool) and each sample in the pool had a unique 6bp barcode. A PCR was performed to incorporate the barcodes before capture to the array.
2.5.1.2 Library preparation and index PCR
Genomic DNA was prepared for high throughput sequencing by following the Illumina ‘Preparing Samples for Multiplexed Paired-‐End Sequencing’ Protocol (part #1005361 Revision B December 2008 available at www.illumina.com). Multiplex reagents were supplied by Illumina as part of the Multiplex Sample Preparation Oligonucleotide Kit (cat# PE-‐400-‐1001).
The Illumina 1005631 protocol outlines nebulization as a method for DNA fragmentation, however this application was modified to use sonication instead. 5µg of genomic DNA was diluted in a total volume of 50µl 1x TE buffer and 150µl of nebulization buffer. The BioRupter sonicator apparatus was assembled and filled with water and ice. Samples were loaded (6 per load) and sonicated for 30 minutes. Fragmented DNA was cleaned with QIAquick PCR purification kit (Qiagen cat # 28106) and 1µl was run on DNA 7500 bioanalyzer chip for size confirmation. The library preparation steps following DNA fragmentation were a) repairing the ends of double stranded DNA fragments; b) adding an A base overhang to the 3’ ends of DNA fragments; c) ligation of DNA to double stranded DNA adapters. A purification step was performed with the QIAquick kit subsequent to steps a, b and c. Ligated DNA templates were size selected (between 250-‐300bp) and then a 6bp index (or barcode) was added via an 18 cycle PCR. After quantification on a spectrophotometer (Nanodrop, Nanodrop Technologies, USA), 12 indexed libraries were pooled together with the same concentration of each library in the pool.
2.5.1.3 Array capture and PCR
Array capture and post capture PCR was performed according to manufacturer’s instructions (Nimblegen Sequence Capture User Guide: Sequence Capture Array Deliver v3.1, available at www.nimblegen.com)
Prior to microarray hybridization, a solution of Cot-‐1 DNA and 1ug of the pooled DNA library was dried down, resuspended in molecular grade water and then incubated at 70°C. Hybridization enhancing oligonucleotides (forward and reverse primers used in post capture PCR, added in excess, to prevent duplex formation between adapter regions of the genomic fragments) and hybridization buffers were added to the re-‐suspended pooled DNA and then denatured at 95°C. Samples were loaded onto a 2.1M sequence capture exome array and hybridized at 42°C for 66-‐72 hours on the Nimblegen hybridization system.
After hybridization, washing and elution of the post-‐captured library was performed; elution at 95°C with water was later replaced with elution with sodium hydroxide at room temperature (Nimblegen Sequence Capture User Guide: Sequence Capture Array Deliver v3.2, available at www.nimblegen.com); both methods were used in this experiment. A final PCR step on the eluate was carried out, where a reaction mix was prepared for 10 reactions per capture. For clean up after PCR, 5 reactions were pooled and added to 1250µl of PBI buffer (QIAquick PCR purification kit), and eluted in a total of 100µl EB buffer.
2.5.1.4 Enrichment qPCR
Enrichment qPCR was performed according to manufacturer’s instructions (Nimblegen Sequence Capture User Guide: Sequence Capture Array Deliver v3.1, available at www.nimblegen.com). This assay determined whether capture was successful and used a standardized set of qPCR SYBR Green assays as internal quality controls for Nimblegen sequence capture experiments (referred to as NSC assays). The genomic loci recognized by the assays were included as capture targets on every human NimbleGen array and comparison by qPCR of
the relative DNA concentrations of the control loci in non-‐captured and captured samples allowed estimation of enrichment of a capture target. There were four NSC assays (NSC-‐0237, NSC-‐0247, NSC-‐0268, NSC-‐0207, refer to manufacturer’s guide for primer sequences) and primers for each assay were diluted to 2um. Each non-‐capture and capture (post PCR) product was diluted to 5ng/µl in PCR grade water to use as qPCR templates. The following volumes of each component were added together to make a qPCR mix for one sample: 5.9µl PCR-‐grade water, 0.3µl NSC 2µm forward primer, 0.3µl NSC 2µm reverse primer, 7.5µl SYBR Green Master (2X), 1µl of 5ng/µl template (non-‐captured product, captured product or positive control genomic DNA templates). The following program was created on the Corbett Rotor-‐gene RT-‐PCR machine: pre-‐incubation 1 cycle 95°C 5 min; amplification 40 cycles 95°C 10 sec; melting curve 1 cycle of 60°C 1 min, 95°C 10 sec and 65°C 1 min; cooling cycle 40°C 10 sec. CT values for all reactions were collected and calculated for replicate reactions. For each different sample and NSC assay, the average CT value of captured template was subtracted from the non-‐captured template. This value was defined as the delta-‐CT. The fold enrichment was calculated for the NSC control locus by raising the PCR efficiency (or E: 1.84 for NSC-‐0237, 1.8 for NSC-‐ 0247, 1.78 for NSC-‐0268, 1.93 for NSC-‐0207) for that assay to the power of delta-‐CT measured for the corresponding control locus. This enrichment analysis was performed for every pool.
2.5.1.5 Pooled DNA library quantification
Prior to clustering and sequencing a sample on the high-‐throughput sequencing machine, the library was quantified by Nanodrop and assayed for size by running 1ul on Agilent 2100 Bioanalyzer with the DNA 7500 chip. The size of the DNA peak was determined by manual inspection of the resulting electropherogram. The molar concentration of the DNA was calculated using the formula below and the library diluted to 10nm for clustering:
where y = the molar concentration of the library (nM); c = concentration of DNA library in ng/µl; x = average molecular weight of a DNA base at 324.5; s = the resultant size of the DNA library. Samples were diluted to 10nM using the formula described.
2.5.2 Nimblegen EZ SeqCap human exome in-‐solution (Phase two)
A total of 75 single samples from coeliac individuals (DNA extracted from either blood or saliva) were prepared for high throughput sequencing and captured with 2.1 million Nimblegen EZ SeqCap human exome in-‐solution probes, covering 26.7Mb and 44.1Mb of the exome for the v1.0 kit and v2.0 kit respectively. Modifications to the protocol include the removal of pre capture PCR and replacing post capture LM-‐PCR with real time qPCR using SYBR green fluorescent dye. This required monitoring cycle-‐to-‐cycle amplification and executing PCR before it reached the amplification plateau. This modified protocol is in Appendix I-‐B where instructions for library preparation, in-‐solution captures and post capture PCR are outlined.
2.5.2.1 Single library quantification with qPCR
Single in-‐solution exome capture libraries were quantified with TaqMan qPCR, developed by Barts and the London Genome Centre. Previous libraries that generated optimum cluster numbers were used as controls. Target cluster numbers per sample were between 350-‐400K per tile with version 3 sequencing chemistry on the Illumina GAIIx. Serial dilutions of 1ul per library and chosen controls were prepared with molecular grade water to give the following concentrations: 20x, 200x, 2000x, 20000x and 200000x. 5.5µl of TaqMan Universal PCR master mix (Applied Biosystems cat #4304437) and TaqMan
Tamra Probes (FAM reporter dye) (Applied Biosystems cat #450025) and 5ul of each triplicate of 2000x, 20000x and 200000x dilutions, plus a water control, were loaded onto a 384 well plate on an automated Biomex FX pre PCR robot, to give a final reaction mix of 10.5µl. The prepared 384 well plate was loaded onto the ABI 7900 HT Fast Real Time PCR system and run at the following cycling times: 50°C for 2 min, 95°C for 10 min, 60 cycles of 95°C for 15 min ad 60°C for 1 min. SDS 2.3 software was used to analyse results at absolute quantification by a ∆CT method.
2.6 Illumina Immunobeadchip genotyping
The entire exome sequencing cohort of 75 samples from phase two of the exome sequencing experiment plus 7728 UK coeliac cases and 8274 UK controls were genotyped on the Immunochip custom chip designed by Illumina. DNA was diluted to 200ng total with molecular grade water in a 96-‐well plate (Thermo Scientific cat #AB-‐0564). Genotyping was performed following the Illumina Infinium HD Ultra User Guide 11328087 Revision B (available at www.illumina.com) at Barts and the London Genome Centre. Genomic DNA was whole genome amplified without PCR, by overnight incubation. After fragmention, precipitation and resuspension, each DNA sample was hybridized to the custom beadchip in a capillary flow-‐through chamber. Non-‐specific DNA was washed away and then stained for single base extension of the oligonucleotides present on the beadchip. As the captured DNA is used as a template, the detected label is incorporated onto the beadchip, and thereby determining the genotype of the sample. All beadchips were analysed on the Illumina iScan housed at the Institute of Health, University College London.
2.7 Fluidigm 48.48 Access Array Integrated Fluidic Circuit technology
Fluidigm Access Array Integrated Fluidic Circuit (IFC) technology was used to for target specific amplification of 26 exome sequencing candidate genes in 2,304 cases and 2,304 controls, equating to three 1,536-‐multipex libraries. DNA