Introductory Next Gen Workshop

Transcription

1 Introductory Next Gen Workshop

2 Workshop Objectives Workshop aimed at those who are new to Illumina sequencing and will provide: - a basic overview of the technology and our service - introduce the types of samples that can be sequenced - provide an overview of sample submission process and web tools for electronic submission Later workshops will focus on library making and bioinformatics

3 What is the Solexa Technology? - Massively parallel sequencing approach resulting in a yield of >1 Gb of sequence data per run (3730xl currently ~77Kb per run) - For an output of 36nts per sequence, this is 4 to 7 million sequences per lane - Utilizes a strategy known as bridge amplification to generate clusters - Clusters are detected and DNA sequence determined via fluorescent nucleotides and a CCD camera -Sequence yield can be doubled using paired-end reads (sequence from both ends of fragment using a paired-end module)

4 How Does it Work? Molecular Biology (libraries, amplification, sequencing by synthesis) Microscopy (laser, prism, objective) Imaging (CCD) Base calls + analyzed data Image Analysis Bioinformatics

5 DNA Preparation Genomic DNA preparation Nebulizer or sonication Start with 1 to 5μg DNA 2% agarose gel Phusion polymerase 200 to 250bp

6 DNA Preparation Sonicator (Biodisruptor) Nebulizer included with DNA prep kits A Hydroshear is also available in the Genomics Core

7 DNA Preparation Genomic DNA Ladder Genomic DNA Ladder Genomic DNA Ladder Nebulizer included with DNA prep kits Sonicator (Biodisruptor) Abel Rosado

8 DNA Preparation Adaptors are ligated to sheared DNA and then size fractionated through a 2% agarose gel Precise sizing (plus/minus 25bp) is more important than yield!

9 Quality Control of Libraries Library length and concentration confirmed using Agilent Bioanalyzer Ladder Genomic DNA Ladder Genomic DNA Small RNA Small RNA (multiplexed) Experimenting with qpcr for cluster Number prediction

10 Library Kits Available from Illumina There are a selection of library kits for different applications - DNA library (genomic, small RNA) - Chromatin Immunoprecipitation (ChIP) sequencing - Small RNA (tag profiling) - Gene expression (tag profiling) - mrna-seq (random cdna) Custom adaptors are used by many individual labs as well (for multiplexing and targeted sequencing) To save money, can make primers (for small RNA) and purchase polymerase separately - other primers can be purchased as a primer only kit - we can provide all Illumina primer sequences

11 New Kits Available from Illumina Just released Multiplexing genomic DNA - kit for multiplexing up to 12 DNA samples per lane using paired-end strategy (product literature on the website) mrna-seq - kit for sequencing random-primed cdnas (mrna-seq) for transcriptome analysis Mate pair library kit - permits longer distances between paired end reads Just released Software upgrades permitting longer reads (up to 75 bp) - kit for 18nts (single and paired-end read) - kit for 36nts (single and paired-end read)

12 Cluster Generation Within the Cluster Station: - Size-selected fragments from library are annealed to complementary primers immobilized on the surface of a flow cell - Multiple rounds of amplification generate clusters of amplicons

13 Cluster Station flow cell Peltier device Within the Cluster Station: - Size-selected fragments from library are annealed to complementary primers immobilized on the surface of a flow cell - Multiple rounds of amplification generate clusters of amplicons flow cell

14 Sequencing Within the Genome Analyzer: - Sequential incorporation of fluorescent nucleotides are detected by a CCD camera - Each cluster is in a fixed location enabling the optics and software to track color changes (sequence) generated at each position - GA II instrument is designed for up to 50nt (36nt standard) with >99% accuracy (new reagents and software extend reads to 50 and 75nts)

15 Sequencing 100 tiles per lane 1 tile 8 lanes per flow cell Within the Genome Analyzer: - Sequential incorporation of fluorescent nucleotides are detected by a CCD camera - Each cluster is in a fixed location enabling the optics and software to track color changes (sequence) generated at each position - GA II instrument is designed for up to 50nt (36nt standard) with >99% accuracy (now extended to 75 nt)

16 How are clusters images? CCD flowcell prism Heating unit objective fluidics laser For each 36 cycle run generate about 1 TB of tiff image files 4 images/tile x 100 tiles per lane x 8 lanes x 36 cycles = 115,200 images! For a paired-end read, image number doubled

17 Basic Data Analysis From the Analysis Pipeline software and dedicated server: - Base calling and measures of read quality such as nucleotide bias, error rates - Basic alignment tools More detailed and project-specific needs are handled by the IIGB Bioinformatics Core - Two full-time programmers for analysis

18 Overview of IIGB Service DNA/RNA preps Library preparation QC Individual labs ~ 1 week - Cluster prep -Sequencing - QC (Illumina software) Genomics Core Service ~ 1 week (36nt single read) Run time will double for PE runs Data analysis (Illumina, Custom analysis) Bioinformatics Core (2 dedicated programmers) Weeks/Months

19 Applications Application Illumina Kit Customer Developed Genomic DNA Transcriptome Whole genome re-sequencing Targeted re-sequencing Digital Gene Expression (tag counting) Transcriptome Sequencing (mrna-seq) ChIP Seq Gene Regulation Small RNA Methylation (Bisulfite Sequencing) DNase1 Hypersensitivity

20 Types of Samples at UCR CHIP Seq Arabidopsis Nucleosome (MNase) & non-nucleosome islands (FAIRE) Plasmodium falciparum Projects under discussion Organelle genome sequencing Microbial genome sequencing Sequencing to identify mutations Small RNA Discovery Arabidopsis Human Citrus Mussa domestica (housefly) Aedes aegypti C. elegans Drosophila Transposon Insertion Sites Drosophila yeast The GA II is a powerful tool for gene expression, small RNAs and genome re-sequencing

21 Customer Project Workflow Meet with Genomics and Bioinformatics Core - Is the project feasible technically and informatically? - What will be the output? - What will it cost? Library preparation (customer supplied) Quality control library - Bioanalyzer (Core personnel) - Sanger sequencing (customers) Establish user account and upload project information Provide physical samples to Genomics Core Receive notification of run completion and pipeline analysis - quality control report, basic alignment scores

22 IIGB Natasha Raikhel (Director IIGB, CEPCEB) Glenn Hicks (Administrator IIGB, Res Cell Biologist B&PS) Jocelyn Brimo (Analyst) Nick Rainsberry (Financial Analyst) Guille Baca (Asst Admin Analyst) David Carter (Academic Coordinator, Microscopy Core) Sonquin Pan (Academic Coordinator, Proteomics Core) Thomas Girke (Assistant Prof, Director, Bioinformatics Core) Tyler Backman (Bioinformatics Analyst) Rebecca Sun (Bioinformatics Analyst) Barbara Walter (Staff Research Associate, Genomics) Clay Clark (Research Assistant, Genomics)

23 HT Sequencing Website