Next-generation sequencing and PBRC. Next Generation Sequencer Applications DeNovo Sequencing...
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Transcript of Next-generation sequencing and PBRC. Next Generation Sequencer Applications DeNovo Sequencing...
Next Generation Sequencer Applications
• DeNovo Sequencing• Resequencing, Comparative Genomics• Global SNP Analysis• Gene Expression Analysis• Methylation Studies• ChIP Sequencing-transcription factors, histones, polymerases• Transcriptome Analysis-splicing, UTRs, cSNPs, nested transcripts• MicroRNA Discovery and quantitation• Metagenomics, Microbial diversity• Copy number variation• Chromosomal aberrations• Gene regulation studies
How many sequence tags* do I need for my gene expression application?
• SAGE/CAGE – 2-5 million mappable• miRNA – 10 million mappable• ChIP Seq—10-20 million mappable• Whole Transcriptome from polyA RNA – 40-50 million mappable• Whole Transcriptome from rRNA depleted - >50 million mappable• Whole Transcriptome for Allele Specific Expression - >>50 million mappable
SOLiD™ 4 generates >1.4 billion mappable sequences/run (2 slides)
Libraries can be multiplexed to decrease the cost/sample according to the application and number of sequences needed.
*For human/mouse sized genomes; smaller organisms require fewer sequence tags.
SAGE Sequencing vs. Microarray
SOLiD v4 Microarray-Illumina Ref 8
Microarray-Illumina Ref 6
Data Points 3.6 million 25,600 45,200
Known and novel transcripts
Known transcripts Known transcripts
Sensitivity 6 logs 3 logs 3 logs
Technical Reproducibility >.99-.999 0.9 0.9
Correlation to Taqman 0.9 0.7-0.8 0.7-0.8
Multiplexing/Barcoding Yes –up to 48 RNA or 96 DNA samples
No No
No background –better for low abundance transcript
detection
Hybridization process creates background signal Hybridization process
creates background signal
RNA quantity 5-10 ug 750 ng 750 ng
16 Sample Experiment Cost
$7200-full service$6100-PI creates library
$3600 $5200
Primary Data Analysis - Images to bases
Tertiary Data Analysis – Experiment Specific
Instrument-specificSequences +Quality values
• Differential expression• Methylation sites• Binding sites• Gene association• Genomic structure
Ref Seq + AlignmentAssembly, De Novo
Secondary Data Analysis – Bases to alignments/contigs
Applications• Tag Profiling• Small RNA
Analysis• Transcriptome
seq.• ChIP-Seq• Methylation
Analysis• Resequencing• De novo
assembly
Algorithms• Eland• Maq• SOAP• Velvet• Newbler• Mapreads• Others …
Run Q
ualit
y
Sam
ple
/Lib
rary
Qualit
y
Dis
covery
Bioinformatics: Geospiza
One or moreData sets
Next-gen sequencing: applications
– Genome analysis: basic and translational research• Genetics of disease – new frontiers• Exome resequencing: confirmation of GWAS• Genome sequence as diagnostic tool• Genetic counseling
– Epigenome analysis: basic research; biomarkers• Analyses of DNA methylation, transcription factors, histone
modifications, non-coding RNA• Epigenomic biomarkers of disease
– Gene expression analysis: basic research; diagnostics & biomarkers• Whole transcriptome: all transcribed sequences in a cell• SAGE analysis: expression of known genes• Small RNA: microRNA as regulators of biology
– Genotype to phenotype: a new frontier• Pathology: systems biology• Diagnosis: data filtering• Personalized Genomic Medicine: Treatment recommendations