Considerations for Illumina library preparation. Henriette O Geen June 20, 2014 UCD Genome Center

Transcription

1 Considerations for Illumina library preparation Henriette O Geen June 20, 2014 UCD Genome Center

2 Diversity of applications De novo genome Sequencing ranscriptome Expression Splice Isoform bundance Genotyping Genome Resequencing Genetic ssociation Metagenomics Exome Sequencing DN Methylation ChIP-Seq Small RN

3 Illumina Workflow Library Preparation Cluster Formation Sequencing Computer nalysis

4 Illumina Sequencing echnology Sequencing By Synthesis (SBS) echnology 3 5 DN ( ug) Library preparation Single Cluster molecule generation array C C G C G G 5 G C C G C C C G C G Sequencing

5 Sequencing workflow Library Construction Cluster Formation Illumina Sequencing Data nalysis

6 Examples of DN input requirements Illumina library prep kit ruseq DN KP DN NEB Ultra Low ruseq ChIP/MeDIP Rubicon hruplex Nextera Kit * Starting material > 100 ng > 10 ng > 5 ng ng 50 pg 50 ng 50 ng DN * Unique protocol using tagmentation : DN is simultaneously fragmented and tagged with sequencing adapters

7 DN library construction Fragmented DN End Repair 5 P OH HO P 5 Blunt End Fragments ailing 5 P P 5 Single Overhang Fragments dapter Ligation DN Fragments with dapter Ends

8 If you can put adapters on it, we can sequence it!

9 Know your sample

10 DN fragmentation Mechanical shearing: NGS BioRuptor Covaris Enzymatic: Fragmentase Chemical

11 Size selection and clean-up using SPRI Beads SPRI = Solid Phase Reversible Immobilization Ratio of SPRI beads/peg solution to sample determines size cut off

12 Optional: PCR-free libraries PCR-free library: OR Library can be sequenced if concentration allows Reduction of PCR bias for eg GC rich regions, especially for metagenomic samples Library enrichment by PCR: Ideal combination: high input and low cycle number

13 Enrichment of library fragments 5 5 PCR mplification

14 HE EVOLUION OF ILLUMIN DPERS ISBELLE HENRY

15 daptors Fragmented DN Regular adaptors + Forward read Reverse read dvantages: Simple Obtain 1-2 reads (F and R) Problems: No multiplexing

16 daptors Fragmented DN Barcoded adaptors + Forward read Reverse read dvantages: Can multiplex Simple Obtain 1-2 reads (F and R) Problems: Cluster detection on the High Seq Lose sequence data in the barcodes

17 daptors Fragmented DN Indexed adaptors + Forward read Index read Reverse read dvantages: Index independent of read -> more data -> no more clustering problems Problems: Need more reagents Index only on one side

18 daptors + Fragmented DN Dual indexed adaptors For 96 reactions Simple index: 96 B adaptors 1 adaptor Dual index: 12 adaptors 8 B adaptors Forward read Index read 1 Index read 2 Reverse read dvantages: Cheaper Indexing information on both sides Problems: B

19 Quantitation & QC methods Intercalating dye methods (PicoGreen, Qubit, etc.): Specific to dsdn, accurate at low levels of DN 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 DN chip allows quantitation of low DN levels qpcr Most accurate quantitation method More labor-intensive Must be compared to a control from a similar library

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

21 Bioanalyzer ChIP options DN1000 High Sensitivity 0.1-5ng/uL

22 Library QC by Bioanalyzer ~ 125 bp Beautiful 100% dapters Beautiful

23 Library QC ~125 bp Examples for successful libraries dapter contamination at ~125 bp

24 Library quantitation by qpcr his step is usually performed by sequencing service center Use amplifying primers corresponding to ends of adapters Use standards of known concentration to generate standard curve of threshold Ct vs. concentration Use conversion factor to deduce concentration of unknown libraries ake library size into consideration! Commercial kits are available Primer 1 Primer 2

25 Examples of RN input requirements Library prep kit mrn (ruseq) Directional mrn (ruseq) NEB ultra directional RN Small RN (ruseq) Ribo depletion (Epicentre) SMRer Ultra Low RN (Clontech) SMR-seq2 Starting material 100 ng - 4 μg total RN 1-5 μg total RN or 50 ng mrn ng mrn or ribo depleted RN 1 μg total RN 1-5 μg total RN 100 pg 10 ng total RN Single cell Single cell

26 Standard RN-Seq library protocol QC of total RN to assess integrity Removal of rrn (most common) mrn isolation rrn depletion Fragmentation of RN Reverse transcription and secondstrand cdn synthesis Ligation of adapters PCR mplify Purify, QC and Quantify

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

28 Strand-specific RN-seq Standard library (non-directional) ntisense non-coding RN Sense transcripts Informative for non-coding RNs and antisense transcripts Essential when NO using poly selection (mrn) No disadvantage to preserving strand specificity

29 Single cell genomics and transcriptomics Cell-to-cell variability (even within same cell type) issue heterogeneity Currently two main methods for single cell transcriptomics Fluidigm C1 system SMR-seq2

30 Krasnow & Stephen R. Quake Nature (2014) doi: /nature13173

31 Deatiled protocol for single cell RN-seq

32 mplicon sequencing Sequencing of amplified regions of interest Common application: 16S/18S small subunit ribosomal RN (SSU rrn) genes as phylogenetic markers Primer 1 Primer 2 OR Standrad library preparation

33 ringe & Rubin (2005) Nature Reviews Genetics 6,

34 hank you!