Lecture 2: Applica:ons for HTS sequencing

Transcription

1 BIT 815: Deep Sequencing Lecture 2: Applica:ons for HTS sequencing Jennifer Schaff Genomic Sciences Laboratory

2 Next genera:on Sequencing Strategies Sequence capture Targeted capture Enrichment capture Exome Linkage peak Epigenome Gene regulalon DNA Methylome BS sequencing Histone profiling Pla.orm / Approach Whole ome & / or 454 Assembly GA/ SOLiD Genome Transcriptome Assembly Combined assembly & analysis Whole genome, known e.g. human Whole genome, de novo e.g. plant Indexed genomes e.g. bacterial QuanLtaLve RNA seq QualitaLve Normalized

3 Applica:ons for HTS Eukaryote Whole Genome Prokaryote Whole Genome Transcriptome Sequencing ChIP seq/methylalon/epigenelcs Expression Tags/Digital Microarrays Metagenomics & Microbial Diversity Mitochondria/ Viruses/ PlasLds/ Plasmids Sequence Capture Targeted Region Small RNA SomaLc VariaLon DetecLon Ancient DNA

4 Applica:ons for HTS Eukaryote Whole Genome Assemble into genomes or transcriptomes Prokaryote Whole Genome Transcriptome Sequencing ChIP seq/methylalon/epigenelcs Gene expression and control Expression Tags/Digital Microarrays InformaLon obtained through Small RNA number of sequences Metagenomics & Microbial Diversity Mitochondria/ Viruses/ PlasLds/ Plasmids Sequence Capture Targeted sequencing SomaLc VariaLon DetecLon Ancient DNA

5 Applica:ons for HTS Eukaryote Whole Genome Assemble into genomes or transcriptomes Prokaryote Whole Genome Transcriptome Sequencing ChIP seq/methylalon/epigenelcs Gene expression and control Expression Tags/Digital Microarrays InformaLon obtained through Small RNA number of sequences Metagenomics & Microbial Diversity Mitochondria/ Viruses/ PlasLds/ Plasmids Sequence Capture Targeted sequencing SomaLc VariaLon DetecLon Ancient DNA

6 Applica:ons for HTS Eukaryote Whole Genome Assemble into genomes or transcriptomes Prokaryote Whole Genome Transcriptome Sequencing ChIP seq/methylalon/epigenelcs Gene expression and control Expression Tags/Digital Microarrays InformaLon obtained through Small RNA number of sequences Metagenomics & Microbial Diversity Mitochondria/ Viruses/ PlasLds/ Plasmids Sequence Capture Targeted sequencing SomaLc VariaLon DetecLon Ancient DNA

7 Transcriptome Sequencing The transcriptome is the set of all RNA molecules one cell, Lssue, organism mrna messenger RNA, contains chemical blue print of proteins and is what most people think of when discussing RNA and transcriptomes rrna RNA component of the ribosome trna transfers a specific aclve amino acid to a growing polypeplde chain other non coding RNA (not translated into a protein), includes snorna, micrornas, sirnas, and others

8 Transcriptome Sequencing The transcriptome is the set of all RNA molecules one cell, Lssue, organism mrna messenger RNA, contains chemical blue print of proteins and is what most people think of when discussing RNA and transcriptomes rrna RNA component of the ribosome trna transfers a specific aclve amino acid to a growing polypeplde chain other non coding RNA (not translated into a protein), includes snorna, micrornas, sirnas, and others

9 Transcriptome Sequencing Full plate sequencing Total Tobacco reads per gene # gene # of genes Tobacco reads/gene # Reads per gene

10 Transcriptome Normaliza:on Image from Evrogen

11 Transcriptome Normaliza:on Images from Evrogen

12 Transcriptome Normaliza:on 500nt Axis in seconds Image from Evrogen Axis in basis

13 Epigene:cs Epigene:cs heritable changes in gene expression (or pheynotype) NOT caused by changes in the underlying gdna sequence When CH Waddington coined the term (1942) the physical nature of genes and their role in heredity was not known; he used it as a conceptual model of how genes might interact with their surroundings to produce a phenotype. Wikipedia hfp://en.wikipedia.org/wiki/epigenelcs The Greek prefix epi implies features that are "on top of" or "in addilon to" genelcs; thus epigene+c traits exist on top of or in addilon to the tradilonal molecular basis for inheritance. Many different molecular mechanisms aclvalon, deaclvalon or modificalon of certain genes (NOT the structure of DNA) includes modificalon of proteins associated with DNA/chromaLn.

14 Epigene:cs EpigeneLc changes are preserved when cells divide, some that apply to research that can be addressed by sequencing: ParamutaLon (methylalon and reg RNAs) ImprinLng (germline, methylalon and histone modificalon) Gene silencing (transcriplonal and post ) X chormosome inaclvalon (dosage compensalon) PosiLon effect Reprogramming RegulaLon of histone modificalons and heterochromaln (applies to some of the above) AddiLonally the progress of carcinogenesis, many effects of teratogens Epigene:c research uses a wide range of molecular biology techniques: chroma:n immunoprecipita:on (large scale ChIP chip and ChIP seq), fluorescent in situ hybridizalon, methylalon sensilve restriclon enzymes, and bisulfite sequencing, and bioinformalcs.

15 Epigene:cs Image from Wikipedia

16 ChIP Seq Accurately survey interaclons between protein, DNA (and RNA) to interpret regulalon events central to many biological processes and disease states. Qualify (and quanlfy) in vivo protein DNA interaclons using the combinalon of chromaln immunoprecipitalon with HTS on a genome wide scale. IdenLfies cistronme of DNA associated proteins. (cis aclng targets, trans aclng factors) Precisely map global binding sites for any protein of interest. Same goal as ChIP chip One of the long term goals ChIP on chip was designed for is to establish a catalogue of (selected) organisms that lists all protein DNA interaclons under various physiological condilons. Wikipedia

17 ChIP Seq Used to analyze protein interaclons with DNA TranscripLon factors ReplicaLon related proteins (ORC) Histones and related modificalons ULlizing this technology with these and other proteins allow the idenlficalon of promoter regions, enhancers, repressors, and silencing elements, insulators, and other sequences that control DNA replicalon

18 ChIP Seq Image from Wikipedia

19 ChIP Seq Immunoprecipita:on (IP) is the technique of precipitalng a protein out of solulon using an an:body that specifically binds to that parlcular protein. Can be used to isolate and concentrate a parlcular protein from a sample containing many thousands of different proteins. Requires that the anlbody be coupled to a solid substrate (e.g. magnelc beads) at some point in the procedure.

20 ChIP Seq Benefits to this over ChIP chip Not limited by pre designing probles Study any immunoprecipitate from any organism with a sequenced genome without any prior assumplons Map in vivo binding sites across the enlre genome Cost Effec:ve: 1/10 1/30th cost of whole genome ChIP chip Robust and Precise Detec:on: Obtain higher signal to noise ralos than array pla.orms, and map binding sites less than 50 bp (Illumina)

21 ChIP Seq

22 ChIP Seq Data Analysis NEED YOUR CONTROLS! This is a biological applicalon, binding sites, lots of steps Need a complete genome to map the sequencing

23 Bisulfite sequencing Chroma:n: a complex of a nucleic acid with basic proteins (such as histones) Histone proteins: are protein spheres that DNA wraps around. Gene expression can be altered if the way DNA is wrapped around it changes. Can happen two ways: Post translalonal modificalon and Addi:on of methyl groups to the DNA MethylaLon of DNA happens mostly at CpG sites and converts the cytosine to 5 methylcytosine Pairs with guanine Highly methylated areas tend to be less transcriplonally aclve (Mechanism not fully understood)

24 Bisulfite sequencing 70% to 80% of CpG cytosines are methylated in mammals CpG occurs with a much lower frequency in the vertebrate genomes than would be expected due to random chance. For example, the human genome is ~42% GC content CpG is expected to occur 0.21 * 0.21 = 4.41% of the Lme. Actual frequency of CpG dinucleoldes in human genomes is 1% less than one quarter of the expected frequency. Methylated cytosines are more susceplble to transilon mutalons Scarano et al. proposed that the CpG deficiency is due to this increased vulnerability (For comparison approximately two out of three (SNPs) are transilons.)

25 Bisulfite sequencing 70% to 80% of CpG cytosines are methylated in mammals (Human DNA has about 80% 90% of CpG sites methylated) Excep:on: CpG islands ~65% unmethylated CG residues (GC rich) Associated with the promoters of 56% of mammalian genes, including all ubiquitously expressed genes. 1 2% of the human genome are CpG clusters, and there is an inverse relalonship between CpG methylalon and transcriplonal aclvity.

26 Bisulfite sequencing Bisulfite treatment (conversion) for the deaminalon of (C)ytosine to (U)racil in DNA has remained the "gold standard" for a number of downstream applicalons developed over recent years to assess DNA methylalon status. Image from Zymo Research Workflow: Denature gdna Bisulfite Conversion DesulfonaLon

27 Bisulfite sequencing Denature double stranded DNA Image from Wikipedia

28 Bisulfite sequencing Image from Dr. Axel Schumaker Bisulfite Conversion and DesulfonaLon

29 Bisulfite sequencing Treatment of DNA with bisulfite converts only unmethylated cytosines to uracil and leaves 5 methylcytosine residues unaffected. Yields single nucleolde resolulon informalon about the methylalon status of a segment of DNA. Can sequence to retrieve this informalon What other informa:on do you need?

30 RNA seq HTS sequencing pla.orms/technologies are used to sequence cdna to get informalon about a sample s RNA content Can use informalon to develop a transcriptome Term is used very ozen for experiments designed to compare transcriptomes (gene expression) between two treatments.

31 RNA seq Microarrays enable interrogalon of thousands of (predetermined) genes at one Lme Data is collected as comparisons between two Lssues

32 RNA seq Microarrays HTS sequencing Pre conceived planning bias No planning bias (can also affect data measurement) Cloning or purchasing of cdnas or oligos No 384 well plates/ libraries QuanLtaLve measurement Low level gene expression can be quanlfied** Always relalve measurement Measure only what is queried $$? (Lme?) Many sources of varialon New transcripts can be detected *sequence and compare profiles at the same Lme $$$? Fewer sources of varialon?

33 RNA seq Transcript populalon Gene A Gene B Gene C Gene D UTR (5 or 3 ) Legend Nebuliza:on Tradi:onal cdna prepara:on for sequencing

34 RNA seq Full plate sequencing Total Tobacco reads per gene # gene # of genes Tobacco reads/gene Microarray type sequencing? # Reads per gene

35 RNA seq Transcript populalon Gene A Gene B Gene C Gene D UTR (5 or 3 ) Legend Nebuliza:on Tradi:onal cdna prepara:on for sequencing

36 RNA seq Transcript populalon Gene A Gene B Gene C Gene D UTR (5 or 3 ) Legend Standard method Nebuliza:on 3 UTR capture 3 UTR capture increases chance of finding rare transcripts Method adapted by Torres et al., Genome Research

37 RNA seq Reads Bases (Mb) Ave length 3' UTR selected 192, Non selected 254, Number of mappable reads Number of genes tagged DistribuLon of tags/ gene 3'UTR/NS 75% 70% 94%

38 RNA seq 3' UTR AF ZM Non selected 84.0% 161,420 Y 179, % 7.7% 14,743 Y 8, % 2.1% 4,051 Y Y 2, % 6.2% 11,912 64, % 100% 192, , % Number of mappable reads

39 RNA seq 3'UTR Non selected # of genes Y Y 12,739 Y 8,531 Y 10,041 Total unique genes 31,311 Number of Maize genes tagged

40 RNA seq 3'UTR Non selected # of genes Y Y 1,573 Y 1,479 Y 1,257 Total unique genes 4,309 Number of A. flavus genes tagged

41 RNA seq Number of reads per gene, both methods #reads #genes 3'UTR #genes NS total % of genes % of genes NS 3'UTR

42 RNA seq Number of reads per gene, both methods #reads #genes 3'UTR #genes NS total % of genes % of genes NS 3'UTR

43 RNA seq Number of reads per gene, both methods #reads #genes 3'UTR #genes NS total % of genes % of genes NS 3'UTR

44 RNA seq Number of reads per gene, both methods #reads #genes 3'UTR #genes NS total % of genes % of genes NS 3'UTR

45 RNA seq Summary (preliminary) of expression analysis through sequencing Data Produc:on Mappable Reads Number of Tagged Genes Distribu:on of Tags 3'UTR (++) ++ NS ++ + (++) +

46 Small RNA The transcriptome is the set of all RNA molecules one cell, Lssue, organism mrna messenger RNA, contains chemical blue print of proteins and is what most people think of when discussing RNA and transcriptomes rrna RNA component of the ribosome trna transfers a specific aclve amino acid to a growing polypeplde chain other non coding RNA (not translated into a protein), includes snorna, micrornas, sirnas, and others Usually under 30nt in length

47 Small RNA HTS applicalons: Discovery of Small RNAs through sequencing Profiling of small RNA regulatory elements Regulatory element comparison between Lssues, treatments, etc.

48 Small RNA Small RNA Sample PreparaLon protocol is used to prepare a variety of RNA species. The protocol takes advantage of the natural structure common to most known microrna molecules. Most mature mirnas have a 5 phosphate and a 3 hydroxyl group as a result of the cellular pathway used to create them. Because of this, the Illumina adapters in this kit are directly, and specifically, ligated to mirnas.