Molecular Biology and Functional Genomic Core Facility

Transcription

1 Molecular Biology and Functional Genomic Core Facility General Presentation Dr Odile Neyret Core Manager Myriam Rondeau Research Assistant Agnès Dumont Research Assistant Institut de recherche clinique de Montréal Room Avenue des Pins ouest Montréal (QC) H2W 1R7

2 Molecular Biology and Functional Genomic Core General Overview Services provided : Sanger sequencing Genotyping Special projects DNA and RNA quality control Next Generation Sequencing (NGS) Accessible devices : 2 Viia7 Q-PCRs Covaris (DNA shearing) 2 Nanodrop Stratalinker

3 Molecular Biology and Functional Genomic Core Services provided : Sanger sequencing Genotyping Special projects DNA and RNA quality control Next Generation Sequencing (NGS) Accessible devices : 2 Viia7 Q-PCRs Covaris (DNA shearing) 2 Nanodrop Stratalinker

4 DNA Sequencing by Sanger dideoxy method Principle : Exploits the ability of DNA polymerase to incorporate 2-3 dideoxynuclotide (ddntp) that terminates the synthesis Technique : 2 steps = Extension (1) & Electrophoresis (2) (1) From a UNIQUE primer, DNA is extended by T7 DNA polymerase at 50 C in PCR device Cycle sequencing fluorescent dyelabeled ddntp +

5 DNA Sequencing by Sanger dideoxy method (2) : Capillary electrophoresis and detection of the fluorescence Sequence can be read up to 1,000 bp depending sample quality. Genetic Analyzer 3500xl -24 capillaries - length 50cm

6 DNA Sequencing : Procedure PCR Product = Amplicon Samples Preparation Plasmid/BAC gdna Removal excess dntp, primers, nucleases & salt ions Sequencing reaction 2h30 min Extension reaction : dntp, ddntp*, T7pol,.. Template 1 sequencing primer Beads Purification 30 min Capillary Electrophoresis 24 samples = 2hrs - 1 plate = 8hrs (O/N) Electrokinetic injection Data Analysis Raw data analysis: level, length, background, general quality

7 DNA Sequencing : Sample submission 1/ Requisition form Primers provided at no cost M13 Forward (-20) M13 Reverse (-27) T7 promotor T7 terminator SP6 upstream EGFP-C EGFP-N pcmv forward BGH reverse Anchor polyt Make sure: Sample name written on the form corresponds to the label on the tube Samples are provided at the correct concentration & volume 1 tube / sample and 1 tube / primer Ask about special condition : ShRNA, BAC, bisulfite treated samples

8 DNA Sequencing : Sample submission 2/ Bring your samples Sequencing days are every Tuesday and Thursday. Be sure to give your samples at last before 8 :30am these days, or the days before. Complete the requisition form and send it by Print a copy of the requisition form and join it with the tubes. Samples could be deliver at IRCM reception desk from Monday to Friday between 8am-4pm. 3/ Results Available : Wednesday am Friday am Warning: Unclaimed samples will be thrown out weekly

9 DNA Sequencing : Softwares Sequencher : Align multiple sequences Compare to ref sequence View chromatograms Find mutations, restriction sites SnapGene : Align multiple sequences Compare to ref sequence View chromatograms => Cloning tool SeqScanner : free download from Intranet Chromas : free download from Internet View chromatograms only

10 Molecular Biology and Functional Genomic Core Services provided : Sanger sequencing Genotyping Special projects DNA and RNA quality control Next Generation Sequencing (NGS) Accessible devices : 2 Viia7 Q-PCRs Covaris (DNA shearing) 2 Nanodrop Stratalinker

11 Genotyping : Procedure Fluorescence-based PCR detection with Genetic Analyzer 3500xl gdna PCR reaction Gene X Applications: - Follow mouse strains - Detect InDel Mutations Multiplexing VIC, FAM, ROX, NED Up to 3 simultaneous PCR reactions, depending on size and fluorophore used Add a ladder in every well Capillary Electrophoresis Results Analysis Fluorescence = High Resolution Capillary length = High Sensitivity and Separation ( pb) GeneMapper software (size, presence, absence)

12 Genotyping : Mouse genotyping PCR Electrophoresis Gene X WT Gene X KO Mouse WT/KO Mouse WT Mouse KO Allele WT 210 bp Allele KO 225 bp

13 Genotyping : InDel detection PCR Electrophoresis Allele WT Allele ins 3 bp insertion WT WT Ins 3bp 155bp 158bp

14 Molecular Biology and Functional Genomic Core Services provided : Sanger sequencing Genotyping Special projects DNA and RNA quality control Next Generation Sequencing (NGS) Accessible devices : 2 Viia7 Q-PCRs Covaris (DNA shearing) 2 Nanodrop Stratalinker

15 Gene analysis by Sanger method : Primer design PCR amplification of exons Sequencing Mutation screening SNP characterization by Q-PCR : Specific probes for targeted SNP Q-PCR with TaqMan chemistry Genotype analysis Special projects In/Del identification by fragment analysis : Fluorescence-based PCR reaction Analyses on Genemapper Methylation analyses by Sanger method : gdna Bisulfite treatment PCR amplification (and cloning) Sequencing => Ask about your projects

16 Molecular Biology and Functional Genomic Core Services provided : Sanger sequencing, Genotyping, Special projects DNA and RNA quality control, Next Generation Sequencing Accessible devices : 2 Viia7 Q-PCRs, Covaris (DNA shearing), 2 Nanodrops Stratalinker

17 Bioanalyzer : Nucleic acids analysis Agilent bioanalyzer 2100 can replace gel electrophoresis analyses of RNA, DNA fragments (shearing, PCR or digestions) when low quantity are involved

18 Bioanalyzer : Nucleic acid analysis RNA Quality : RIN (RNA Integrity Number) evaluation RNA degradation RNA contamination

19 Bioanalyzer : Nucleic acid analysis DNA Quality : NGS : shearing validation NGS : Library validation QC (size, quantity) Other projects : PCR amplifications or digestions

20 Bioanalyzer : Nucleic acid analysis The kits available at the core are : RNA Pico kit : Eucaryote or Procaryote total RNA - 50 to 5,000 pg/µl High Sensitivity DNA kit : Fragments from 50 to 7000 bp - Quantity 5 to 5,000 pg/µl Only 1µl of sample loaded

21 Molecular Biology and Functional Genomic Core Services provided : Sanger sequencing Genotyping Special projects DNA and RNA quality control Next Generation Sequencing (NGS) Accessible devices : 2 Viia7 Q-PCRs Covaris (DNA shearing) 2 Nanodrop Stratalinker

22 Next Generation Sequencing NGS = 1 to 3 Gb (vs Sanger Sequencing = up to 1,000 bp) RNA Seq => transcriptome DNA Seq Epigenome methylation rrna depletion mrna enrichment Whole Genome ChIP Targeted regions Exomes Custom Ribosomal Profiling Small RNA Custom work depending on : - Project - Quantity of starting material

23 Next Generation Sequencing : Workflow DNA/RNA QC bioanalyzer 150 to 600 bp Library Prep Quantification & Quality Control bioanalyzer Q-PCR Ligate Adapter with Index for multiplexing Pool Multiplex in 1 lane Sequencing Illumina HiSeq 2500 Platform Results Analysis Bio-informatics service available : Virginie Calderon

24 Next Generation Sequencing : Workflow Illumina HiSeq 2500 Platform SR/PE SR/PE PE only 50 cycles 7 cycles

25 Next Generation Sequencing : Sample Requisition Note: Samples are processed only if the QC is validated DNA/RNA Seq ng to 1 µg / sample, - Extraction with column or magnetic beads only ChIP Seq - 1 to 10 ng / sample, - Preparation + validation of enrichment

26 Molecular Biology and Functional Genomic Core Services provided : Sanger sequencing Genotyping Special projects DNA and RNA quality control Next Generation Sequencing (NGS) Accessible devices : 2 Viia7 Q-PCRs Covaris (DNA shearing) 2 Nanodrop Stratalinker

27 Q-PCR : Procedure 2 Q-PCRs from LifeTech : Viia7-96 and ViiA hrs time slot reservations: - starting at odd hours 9:00am/11:00am/1:00pm - Must also reserve on weekends Software= QuantStudio can be download for free from the LifeTech website (Mac OS, Windows) Must use plates and optical films from LifeTechnologies Can use MasterMix of your choice

28 Molecular Biology and Functional Genomic Core Services provided : Sanger sequencing Genotyping Special projects DNA and RNA quality control Next Generation Sequencing (NGS) Accessible devices : 2 Viia7 Q-PCRs Covaris (DNA shearing) 2 Nanodrop Stratalinker

29 Covaris : DNA Shearing E220 instrument with focused acoustic technology and refrigerated bath => consistent and reproducible shearing Different protocols depending on application : - gdna shearing for NGS and library construction - high or low cell chromatin shearing for ChIP Analysis Vials recommended by Covaris available at the core facility : - MicroTUBES with snap-cap (6x16 mm) 130µl - MilliTUBES 12x12 mm 1ml

30 Molecular Biology and Functional Genomic Core Services provided : Sanger sequencing Genotyping Special projects DNA and RNA quality control Next Generation Sequencing (NGS) Accessible devices : 2 Viia7 Q-PCRs Covaris (DNA shearing) 2 Nanodrop Stratalinker

31 Self-service Instrumentation : 2 Nanodrop 2000 : Nucleic acid and protein quantification & quality control No bacterial culture measurement in the core => Each user must provide their own materials (blank solution, pipettes, tips, Kimwipes, marker...) Stratalinker 2400 : Crosslink DNA or RNA on membranes => No UV irradiation of cells, or colony screening