Illumina Next Generation Sequencing Data analysis

Transcription

1 Illumina Next Generation Sequencing Data analysis Chiara Dal Fiume Sr Field Application Scientist Italy 2010 Illumina, Inc. All rights reserved. Illumina, illuminadx, Solexa, Making Sense Out of Life, Oligator, Sentrix, GoldenGate, GoldenGate Indexing, DASL, BeadArray, Array of Arrays, Infinium, BeadXpress, VeraCode, IntelliHyb, iselect, CSPro, GenomeStudio, Genetic Energy, HiSeq, and HiScan are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.

2 Illumina Sequencing Workflow Sample Prep Analysis Instrumentation Primary Analysis Secondary Analysis 2

3 Illumina Sequencing Workflow Outcomes Clusters Images/TIFF files Sample Prep Analysis Instrumentation Primary (DNA Library) Intensities Basecalling Secondary Alignments 3

4 4 Primary Analysis Processes

5 Primary Data Analysis Components Instrument Control Software Provides a graphical interface while running the instrument Real Time Analysis (RTA) Component within the Instrument Control Software that monitors the run s progress, optimizes run conditions and provides run-time quality statistics Off-Line Base Caller (OLB) Provides the option to perform data analysis off-line Firecrest (image analysis) Instrument Control Software RTA CIF/files Primary Analysis Bustard (base calling) Off-Line Basecaller CASAVA Secondary Analysis bcl Samplesheet.csv CASAVA Build GenomeStudio or third party apps. 5

6 6

7 Cluster Detection Algorithm Maximum Threshold Cluster 7

8 Multi-Cycle Detection of Cluster Positions Better resolution of some overlapping clusters e.g. when two overlapping clusters are the same base in cycle 1 Cluster detection is performed over 4 cycles CYCLE 1 G channel Difficult to resolve overlapping clusters in one cycle only, when there are overlapping clusters of the same base G channel CYCLE 2 C channel By detecting cluster positions in multiple cycles there is a better chance of resolving overlapping clusters since they are less likely to be of the same base in multiple cycles 8

9 9 Base Calling Algorithm

10 Base calling Algorithms: Base calling and quality filtering Corrected Intensity C A C G T I A I B A C G T Filter clusters by signal purity Second-lowest CHASTITY at least 0.6 in first 25 cycles Looks at more cycles Allows one bad cycle Better error rate for given yield C I A I A I B CHASTITY Formula 10

11 SAV software monitoring run quality 11

12 12 12 Secondary Analysis

13 What is CASAVA? Consensus Assessment of Sequence And VAriation (CASAVA) is a Linux application designed to: Demultiplex samples Align reads Call SNPs Find indels Count expression level for exons, genes and splice junctions in case of RNA-seq runs Primary Analysis CASAVA s output is a folder structure (called a CASAVA Build ) ready for import into GenomeStudio for visualization and further analysis Gerald (Eland v2) Secondary SNP and Analysis Multiple *_export.txt + other files script for multiplexed run Indel Calls Counting For mrna CASAVA Build Data Visualization 13

14 CASAVA 1.8 BCL converter Converter part of CASAVA Generate fastq using ASCII offset of 33 Perform demultiplexing Data analysis on a per Sample basis Data manipulation and sharing greatly facilitated Common data formats (BAM and FastQ) Easier to handle and share Lots of other software available that work with BAM/FastQ 14

15 Secondary and Tertiary Analysis The biological visualization RNA-Seq: Tuxedo suite, Bioconductor, Third party tools MetaGenomics NextGEne, CLCbio, Megan SNP & Indel Detection ELAND/CASAVA, BWA/SAMTools, DeNovo Assembly Abyss, Velvet, SoapDenovo 15

16 Burrows-Wheeler Aligner BWA aligns relatively short nucleotide sequences against a long reference sequence such as the human genome. BWA has two major components, one for read shorter than 150bp and the other for longer reads. Bwa-short: algorithm that works for query sequences shorter than 150bp BWA-SW: algorithm that works for longer sequences up to around 100kbp. BWA Main features: Gapped alignment and paired-end mapping Accurate, fast and lightweight BWA-SW component is able to find chimera. 16

17 Sequence Alignment/Map SAM is a generic format for storing large nucleotide sequence alignments. SAM main features: Flexibility: store all the alignment information generated by various alignment tools Simplicity: easily generated by alignment programs or converted from existing alignment formats Compact in file size Allows the file to be indexed by genomic position to efficiently retrieve all reads aligning to a locus. SAM Tools provide various utilities for manipulating alignments in the SAM format, including sorting, merging, indexing and generating alignments in a per-position format. 17

18 The "Tuxedo Suite : Secondary analysis applications for RNA-seq Aligning Short Reads to References with Bowtie, Tophat, and Cufflinks Provide directions on how to convert Illumina sequence output to: Measures of differential expression Discover splice forms Bowtie Tuxedo suite Cufflinks TopHat 18

19 19 Thank you