Introduction to the MiSeq

Transcription

1 Introduction to the MiSeq 2011 Illumina, Inc. All rights reserved. Illumina, illuminadx, BeadArray, BeadXpress, cbot, CSPro, DASL, Eco, Genetic Energy, GAIIx, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, Infinium, iselect, MiSeq, Nextera, Sentrix, Solexa, TruSeq, VeraCode, the pumpkin orange color, and the Genetic Energy streaming bases design are trademarks or registered trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.

2 MiSeq Sequencing Workflow Amplicons Clones gdna Library Prep 1.5 hours (15 minutes hands-on) Amplification thru Sequencing 4.5 hours (20 minutes hands-on) Alignment and Variant Calling 2 hours (fully automated) 08:00:00 Sample-to-Answer in as Little as Eight Hours* 2 *1x36bp run 3 hr sequencing

3 Simplified Sample Prep using Nextera Technology No separate fragmentation is required Tag with Enzyme mix PCR (polishes fragment ends and incorporates optional index) 3

4 TruSeq Sample Preparation MiSeq can also sequence libraries made using TruSeq Sample Preparation Kits DNA RNA 4

5 Onboard Cluster Generation Bind single DNA molecules to surface Amplify on surface Form clusters ~1000 molecules per ~ 1 um cluster 5

6 Images are Collected for each Nucleotide in each Cycle Clusters are imaged using LED and filter combinations specific for each fluorescently labeled nucleotide More information about SBS technology can be found at our website After imaging is complete for one section (called a tile) the flow cell is moved to the next tile and the process is repeated, similarly to that of Genome Analyzer Imaging for 1 cycle takes about ~3 minutes, including focusing routines A image C image G image T image 6

7 MiSeq Reagent Cartridge MiSeq uses a preloaded single use reagent cartridge Provides Walkaway automation All of the reagents required for cluster generation, sequencing and paired end chemistry are contained within the cartridge Users can load a single library or a pool of indexed libraries into the reagent cartridge for sequencing Radio-Frequency Identification (RFID) allows for positive consumable tracking 7

8 MiSeq Flow Cell MiSeq uses a single lane flow cell Samples are applied to the flow cell from the reagent cartridge sample tube Optimized microfluidics and smaller imaging area allow for faster cycling times Radio-frequency identification allows for positive consumable tracking Recommended cluster density for the MiSeq is 800,000 clusters/mm2 Pin Positions 8

9 9 Simple Reagent and Flow Cell Loading

10 Simple Reagent and Flow Cell Loading 10

11 Simple Reagent and Flow Cell Loading Load flow cell 11

12 Simple Reagent and Flow Cell Loading Flow cell holder ensures proper alignment of the flow cell No extra gaskets required 12

13 MiSeq Control Software Touch screen Graphical User Interface Oversized buttons make on-screen navigation easy Starting a run only takes a few clicks Short video clips demonstrate proper loading of flow cell and reagents 13

14 MiSeq Workflows Workflow for a run is specified in the Sample Sheet Additional Information about the Sample Sheet can be found in MiSeq User Guide Workflow Type Description Output Resequencing DNA sequences are aligned against a reference Aligned reads in BAM format, sequence and variants are noted variant calls Amplicon De novo Assembly Sequencing of PCR amplicons from probes targeting particular genome positions (up to ~384 loci from up to ~96 samples) Assembly of small (< 20MB) genome from reads without the use of a genomic reference Aligned reads in BAM format, variant calls Contigs in FASTA format Small RNA Sequencing of mirna Adapter-trimmed aligned reads in BAM format, reports on the relative abundance of each record Metagenomics 16S rrna sequencing FASTQ and Bacterial classification of sequences 14

15 Primary Data Analysis On MiSeq MiSeq Control Software (MCS) does primary image analysis and produces bases calls and quality scores MCS displays real time run metrics Number of cycles Total run time remaining Cluster density Tile intensity % of clusters that pass filtering (PF rate) Estimated yield of the run Quality score distribution per cycle Run metrics can be monitored remotely via Sequence Analysis Viewer (SAV) 15

16 Visualization of run metrics in MCS 16

17 Secondary Analysis Summarized with MiSeq Reporter All workflows give a general summary (see below) and workflow specific output Secondary analysis is complete within 2 hours of the run finishing 17

18 Resequencing Sample Report Coverage and error Scope of view Zoom in/out Q score SNP/indel + annotation Table of samples/ variants 18

19 Small RNA Sample Report Top 10 most abundant species Distribution of RNA species Tabular information 19

20 What is BaseSpace? BaseSpace is Illumina s genomic cloud computing environment Eliminates need for onsite storage and computing Enables Web-based data management and analysis Provides tools for collaboration and sharing Available GLOBALLY for Illumina and non-illumina customers BaseSpace 20

21 BaseSpace Data Analysis Options for MiSeq Data Illumina Experiment Manager: Define analysis parameters Local Analysis MiSeq Sequencing Results.bcl files MiSeq Reporter Amplicon De Novo Assembly Library QC Metagenomics Resequencing Small RNA Cloud-Based Analysis BaseSpace Amplicon De Novo Assembly Library QC Metagenomics Resequencing Small RNA Third Party Software Secure Data Storage Data Sharing 21

22 Benchmark comparison 22

23 23 Thank you!