Introduction to Illumina NGS technology, Library ...dors.weizmann.ac.il/.../IntroductiontoNGS_libraries... · Introduction to Illumina NGS technology, Library preparation and Mars-seq

Introduction to Illumina NGS technology, Library

preparation and Mars-seq

Hadas Keren-Shaul Advanced Sequencing Technologies (Sandbox), LSCF

Introduction to Deep Sequencing Analysis course June 2017

LSCF

Sandbox A Playground for Genomic Research and

Science Innovation

• Open 24/7

• Access to Weizmann trained users

Sandbox lab, Levine building, Room 202

Genomic technologies are greatly advancing Biology and Medicine

How do we get access to these technologies?

How do we share the technological developments in Weizmann?

The Interface between cutting edge technologies developed in individual labs to the entire community of Weizmann

• Standardizing custom genomic protocols

• Affordable, accessible to many users

• Hands-on workshops

• Quality assured equipment and consumables

• Troubleshooting and guidance

Bringing advanced genomic technologies to Weizmann scientists

Sandbox Vision

Weizmann Genomic

Innovation Sandbox

Weizmann scientists

NGS – Next Generation Sequencing

The research tool to study biological systems with unprecedented throughput, scalability, and speed

Broad range of applications: Sequence whole genomes

Zoom in to deeply sequence target regions

Utilize RNA sequencing to discover RNA variants and splice sites, or quantify mRNAs for gene expression analysis

Analyze genome-wide methylation or DNA-protein interactions

Study microbial diversity in humans or in the environment

NGS rapidly advances over the years

Cost for genome sequencing has drastically declined

Timeline and Comparison of Commercial HTS Instruments

Jason A. Reuter, Damek V. Spacek, Michael P. Snyder Molecular Cell Volume 58, Issue 4, Pages 586-597 (May 2015) DOI: 10.1016/j.molcel.2015.05.004

Developments in high throughput sequencing

PacBio Sequel

The NGS Workhorse - Illumina Sequencing By Synthesis

A. Library preparation

Illumina, Sequencing By Synthesis

B. Cluster Amplification

Illumina, Sequencing By Synthesis

C. Sequencing

https://youtu.be/fCd6B5HRaZ8

Illumina, Sequencing By Synthesis

D. Alignment and Data Analysis

https://youtu.be/fCd6B5HRaZ8

What should I know before sequencing?

• Library quality and quantity

• Type of protocol used

• Type of kit used

• How many bp to read:

rd1, rd2, index1 (i7), index2 (i5)

• Run definitions can be made in Illumina website, basespace

Illumina library construction

Illumina sequencing libraries

P5 P7 Read1 Read2

i5

i7

Insert to sequence

i7

i5

Illumina sequencers

Production scale sequencers

Illumina sequencers

Benchtop sequencers

In the Sandbox - NextSeq, Illumina

• Desktop sequencing machine, fast, flexible, high-throughput

• Independent, easy and accessible sequencing 24/7

• Nextseq training • Detailed run protocols • A downstream analysis pipeline, generated by LSCF

bioinformatics, for immediate demultiplexing of samples • Used daily by many Weizmann labs

RNA-Seq - Method of choice to study Gene Expression

Identification of novel transcripts

Less background noise

Greater dynamic range for detection

How to perform RNA-seq?

Many different methods for library preparation

Strand specific RNA-seq methods – which DNA strand corresponds to the sense strand of RNA

Wiley Interdisciplinary Reviews: RNA Volume 8, Issue 1, 19 MAY 2016 DOI: 10.1002/wrna.1364

How to perform RNA-seq?

Sequencing on DNA molecules instruments: Capture RNA molecules

Convert RNA to cDNA with defined size range

Add adapter sequences on the cDNA ends for amplification and sequencing

Fragmentation – size limitation of sequencing platform

Add 5’ and 3’ adapters

RNA-seq guidelines

RNA extraction method needs to be calibrated per sample used – kits, beads, Trizol, ec.

Selection of Poly(A)+ transcripts – beads, primers in RT

rRNA depletion – for non-poly(A) RNAs (prokaryotic mRNAs, fragmented mRNAs from FFPE)

RNA Input for library preparation

RNA quality and purity • Measure UV absorption- Nanodrop

• RNA has a maximum absorption at 260nm

• A260/280 – level of protein contamination in the sample. Pure RNA =2.1. Acceptable: 1.8-2.0

• A260/230 – level of salt / organic compounds contamination (guanidine salts and phenols, used in RNA isolation protocols). >1.5

• UV absorbance depends on pH of RNA solution.

• Inaccurate under 20 ng/ml (Qubit)

RNA Input for library preparation

RNA integrity

Degraded RNA will not perform well in dowsntream applications!

Run the on a 1% agarose gel -

28S rRNA band should be ~2-fold 18s

Equal intensity indicates some degradation

mRNA rRNA

rRNA

Higher molecular weight bands – can indicated DNA contamination

Smearing below rRNA indicates poor RNA quality

RNA Input for library preparation

RNA integrity

Run RNA on a Bioanalyzer / TapeStation-

RIN – RNA Integrity Number – quality score for total RNA

Mars-seq – Scalable and sensitive RNA-seq

• Library generation for 3’ RNA seq

• Developed in the lab of Ido Amit

• Low input material (1 ng of RNA)

• Stable, suitable for inexperienced users

• Suitable for a wide variety of species and applications (sorted cells, frozen tissues, etc.)

• Ultra low cost due to custom made reactions

• Simple and efficient due to early pooling of samples

• RNA data < 1 week

• A detailed quality control scheme for library evaluation at different steps prior to sequencing

Jaitin, Kenigsberg, Keren-Shaul et al., Massively Parallel single cell RNA-seq. Science 2014

Library construction- Day 1

Step 1: Reverse transcription

Step 4: Second strand synthesis

3’ 5’ An

NT20-UMI-barcode-partial rd2-1-T7 promoter 3’ 5’

Step 2: Sample pooling A

NT20-UMI-barcode-partial rd2rev-T7 promoter 3’ 5’ 5’ 3’

Step 5: In Vitro Transcription

Un-UMI-barcode-partial rd2rev 3’ 5’

RNA

cDNA

2nd strand

Legend:

Step 3: Exonuclease I

5' –T7 promoter-Illumina sequences XXXXXXX NNNNNNNN TTTTTTTTTTTTTTTTTTTTN 3'

BC-7bp UMI-8bp

aRNA

Un-UMI-barcode-partial rd2rev 3’ 5’

Step 7: RNA Fragmentation

Step 8: RNA/ssDNA ligation

P5_rd1 forward

primer

Step 10: Amplification + Illumina primers addition by nested PCR

Step 9: Reverse transcription

Step 6: DNaseI

OH OH

OH

v Un-UMI-barcode-partial rd2rev 3’ 5’ 3’ 5’ partial rd1rev

Un-UMI-barcode-partial rd2rev 5’ 3’

3’ 5’ partial rd1 primer

P7_rd2 reverse

primer

Library ready for Illumina sequencing

OH Un-UMI-barcode-partial rd2rev 5’

partial rd1

P5 P7

5’ 3’ partial rd2

Library construction- Day 2

Is my library good?

• QC1, QC2, QC3

• Qubit value – ~1-10 ng/ml

• TapeStation profile

Sequencing of Mars-seq libraries

• NextSeq® 500 High Output v2 Kit (75 cycles)

• 75 rd1, 15 rd2, no index

• Pooling up to 80 samples in one run

P5 P7 Read1 Read2

Insert to sequence

Read to align to genome

Read 2 to obtain cell and molecule barcodes

Mars-seq Workshop

• 3 days hands-on workshop

• Standard RNA material

• 1 representative per lab

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Thank you for listening

Happy Sequencing