High Throughput Sequencing the Multi-Tool of Life Sciences. Lutz Froenicke DNA Technologies and Expression Analysis Cores UCD Genome Center

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1 High Throughput Sequencing the Multi-Tool of Life Sciences Lutz Froenicke DNA Technologies and Expression Analysis Cores UCD Genome Center

2 DNA Technologies & Expression Analysis Cores HT Sequencing (Illumina & PacBio) Illumina microarray (expression analysis, genotyping) consultations introducing new technologies to the campus shared equipment teaching (workshops)

6 Hi-C Lieberman-Aiden 2010

7 Hi-C Lieberman-Aiden 2010

8 Dovetail Sequencing (Putnam 2015)

9 Bisulfite Conversion METHYL-Seq

10 ChIP-Seq Chromatin Immuno Preciptation DNA/Protein interactions e.g. transcription factors

11 DRIP-Seq R-loops: three-stranded structure Genome-wide mapping of RNA-DNA hybrids

12 Complementary Approaches Illumina Still-imaging of clusters (~1000 clonal molecules) PacBio Movie recordings fluorescence of single molecules Short reads - 2x300 bp Miseq Repeats are mostly not analyzable High output - up to 100 Gb per lane Up to 60 kb, N50 21 kb spans retro elements up to 1,3 Gb per SMRT-cell High accuracy ( < 0.5 %) Error rate 15 % Considerable base composition bias Very affordable De novo assemblies of thousands of scaffolds No base composition bias Costs 5 to 10 times higher Near perfect genome assemblies

13 Genome Resequencing De novo genome Sequencing SNPs, Indels CNVs Rearrangements Metagenomics RNA-seq Gene Expression Splice Isoform Abundance High Throughput Short Read Sequencing: Illumina Exome Sequencing DNA Methylation ChIP-SEQ 3D Organization Genotyping Small RNA

CNVs Long Read Sequencing PacBio Metageno mics Exome

14 Genome Resequencing Rearrangements De novo genome Sequencing RNA-seq Gene Expression Splice Isoform Abundance SNPs, Indels CNVs Long Read Sequencing PacBio Metageno mics Exome Sequencing DNA Methylation ChIP-SEQ 3D Organization Genotyping Small RNA

15 Illumina sequencing workflow Library Construction Cluster Formation Sequencing Data Analysis

16 Fragmentation Mechanical shearing: BioRuptor Covaris Enzymatic: Fragmentase, RNAse3 Chemical: Mg2+, Zn2+ DNA, RNA DNA, RNA RNA

17 DNA library construction Fragmented DNA End Repair 5 P OH HO P 5 Blunt End Fragments A Tailing 5 P A A P 5 Single Overhang Fragments T T Adapter Ligation DNA Fragments with Adapter Ends

18 Enrichment of library fragments 5 5 PCR Amplification

0 ug) Library preparation Single Cluster molecule

19 Illumina Sequencing Technology Sequencing By Synthesis (SBS) Technology 3 5 DNA ( ug) Library preparation Single Cluster molecule generation array A C T C T G C T G A A G 5 T G C T A C G A T A C C C G A T C G A T Sequencing

20 TruSeq Chemistry: Flow Cell 8 channels Surface of flow cell coated with a lawn of oligo pairs

21 Sequencing 1.6 Billion Clusters Per Flow Cell 20 Microns 100 Microns 21

22 Sequencing 100 Microns 22

23 Patterned Flowcell

24 Hiseq 3000: 478 million nanowells per lane

28 What will go wrong? cluster identification bubbles synthesis errors:

29 What will go wrong? synthesis errors: Phasing & Pre-Phasing problems

30 HS2500 PE250 - Q20

31 Intensities - HS3K PE100

32 HS3K PE150

36 FASTQC

37 FASTQC - Nextera

38 FASTQC

39 FASTQC

40 FASTQC

41 FASTQC

42 FASTQC

43 FASTQC

44 FASTQC

45 FASTQC

47 If you can put adapters on it, we can sequence it! single-stranded Adapter Ligation

48 Optional: PCR-free libraries PCR-free library: OR if concentration allows Reduction of PCR bias against e.g. GC rich or AT rich regions, especially for metagenomic samples Library enrichment by PCR: Ideal combination: high input and low cycle number; low-bias polymerase

49 Quantitation & QC methods Intercalating dye methods (PicoGreen, Qubit, etc.): Specific to dsdna, accurate at low levels of DNA Great for pooling of indexed libraries to be sequenced in one lane Requires standard curve generation, many accurate pipetting steps Bioanalyzer: Quantitation is good for rough estimate Invaluable for library QC High-sensitivity DNA chip allows quantitation of low DNA levels qpcr Most accurate quantitation method More labor-intensive Must be compared to a control

50 Library QC by Bioanalyzer Predominant species of appropriate MW Minimal primer dimer or adapter dimers Minimal higher MW material

51 Library QC by Bioanalyzer ~ 125 bp Beautiful 100% Adapters Beautiful

52 Library QC ~125 bp Examples for successful libraries Adapter contamination at ~125 bp

54 Recommended RNA input Library prep kit mrna (TruSeq) Directional mrna (TruSeq) Apollo324 library robot (strand specific) Small RNA (TruSeq) Ribo depletion (Epicentre) SMARTer Ultra Low RNA (Clontech) Ovation RNA seq V2, Single Cell RNA seq (NuGen) Starting material 100 ng 4 μg total RNA 1 5 μg total RNA or 50 ng mrna 100 ng mrna 1 μg total RNA 1 5 μg total RNA 100 pg 10 ng 10 ng 100 ng

55 Standard RNA-Seq library protocol QC of total RNA to assess integrity Removal of rrna (most common) mrna isolation rrna depletion Fragmentation of RNA Reverse transcription and secondstrand cdna synthesis Ligation of adapters PCR Amplification Purify, QC and Quantify

56 18S (2500b), 28S (4000b)

58 RNA integrity <> reproducibility Chen et al. 2014

59 Considerations in choosing an RNA-Seq method Transcript type: - mrna, extent of degradation - small/micro RNA Strandedness: - un-directional ds cdna library - directional library Input RNA amount: ug original total RNA - linear amplification from ng RNA Complexity: - original abundance - cdna normalization for uniformity Boundary of transcripts: - identify 5 and/or 3 ends - poly-adenylation sites - Degradation, cleavage sites

60 Is strand-specific information important? Standard library (non-directional) antisense sense Neu1

61 Strand-specific RNA-seq Standard library (non-directional) Antisense non-coding RNA Sense transcripts Informative for non-coding RNAs and antisense transcripts Essential when NOT using polya selection (mrna) No disadvantage to preserving strand specificity

62 RNA-seq for DGE Differential Gene Expression (DGE) 50 bp single end reads 30 million reads per sample (eukaryotes) 10 mill. reads > 80% of annotated genes 30 mill.. reads > 90% of annotated genes 10 million reads per sample (bacteria)

63 Molecular indexing for precision counts

64 Molecular indexing for precision counts

65 Other RNA-seq Transcriptome assembly: 300 bp paired end plus 100 bp paired end Long non coding RNA studies: 100 bp paired end million reads Splice variant studies: 100 bp paired end million reads

66 RNA-seq cheap and dirty - 3 Tag-sequencing - Micro-array-like data - Quant-Seq (coming soon) - Brad-Seq (Townsley 2015)

67 RNA-seq targeted sequencing: - Capture-seq (Mercer et al. 2014) - Nimblegen and Illumina - Low quality DNA (FFPE) - Lower read numbers 10 million reads - Targeting lowly expressed genes.

68 RNA-seq reproducibility Two big studies multi-center studies (2014) High reproducibility of data given: - same library prep kits, same protocols - same RNA samples - RNA isolation protocols have to be identical - robotic library preps?

69 THIRD GENERATION DNA SEQUENCING Single Molecule Real Time (SMRT ) sequencing Sequencing of single DNA molecule by single polymerase Very long reads: average reads over 8 kb, up to 30 kb High error rate (~13%). Complementary to short accurate reads of Illumina

70 70 nm aperture Zero Mode Waveguide

71 Damien Pelt

First Sequencing of CGG-repeat Alleles in Human Fragile X Syndrome using PacBio RS Sequencer Paul Hagerman,

Single-molecule sequencing of pure CGG array, - first for disease-relevant allele. Loomis et al.

Direct genomic DNA sequencing of methyl groups, - direct epigenetic sequencing (paper under review).

72 First Sequencing of CGG-repeat Alleles in Human Fragile X Syndrome using PacBio RS Sequencer Paul Hagerman, Biochemistry and Molecular Medicine, SOM. Single-molecule sequencing of pure CGG array, - first for disease-relevant allele. Loomis et al. (2012) Genome Research. - applicable to many other tandem repeat disorders. Direct genomic DNA sequencing of methyl groups, - direct epigenetic sequencing (paper under review). Discovered 100% bias toward methylation of 20 CGGrepeat allele in female, first direct methylated DNA sequencing in human CGG disease. 36 CGG 95 DoD STTR award with PacBio. Basis of R01 applications. C A G T Nucleotide position

73 Iso-Seq Pacbio Sequence full length transcripts no assembly High accuracy (except very long transcripts) More than 95% of genes show alternate splicing On average more than 5 isoforms/gene Precise delineation of transcript isoforms ( PCR artifacts? chimeras?)

74 C 1 Single cell capture

75 10X Genomics

77 25x linked read coverage

78 60 kb deletion

83 Thank you!

High Throughput Sequencing the Multi-Tool of Life Sciences. Lutz Froenicke DNA Technologies and Expression Analysis Cores UCD Genome Center

High Throughput Sequencing the Multi-Tool of Life Sciences Lutz Froenicke DNA Technologies and Expression Analysis Cores UCD Genome Center Complementary Approaches Illumina Still-imaging of clusters (~1000