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 2018

LSCF

SandboxA 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

InnovationSandbox

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

PacBioSequel

The NGS Workhorse - Illumina Sequencing By Synthesis

A. Library preparation

Illumina, Sequencing By Synthesis

B. Cluster Amplification

Illumina, Sequencing By Synthesis

C. Sequencing

Illumina, Sequencing By Synthesis

D. Alignment and Data Analysis

https://youtu.be/fCd6B5HRaZ8

Illumina library construction

Illumina sequencing libraries

P5 P7Read1 Read2

i5

i7

Insert to sequence

i7

i5

Paired End (PE) vs. Single read (SE)

Single end (SE) – for each cDNA fragment only one end is readPaired end (PE) – the cDNA fragment is read from both ends

Paired end sequencing

Enables both ends of the DNA fragment to be sequenced Facilitates detection of genomic rearrangements and

repetitive sequence elements, as well as gene fusions and novel transcripts

Better alignment of the reads, especially across difficulty to sequence, repetitive regions of the genome

What should I know before sequencing?

• Library quality and quantity

• Type of library prep method used

• PE or SR?

• How many bp to read:

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

• Run definitions can be made in Illumina website, basespace

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?

Most RNA-seq experiments are based on 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

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

Selection of polyA+ transcripts

The most common application of RNA-seq

In Eukaryotic organisms, most protein coding RNAs (mRNA) contain a poly(A) tail

Technical convenience for enrichment of poly(A)+ transcripts from total cellular RNA (1-5%)

Beads coated with polyT or oligo-dT priming for Reverse transcription (possible 3’ bias)

In non polyadenylated RNAs, such as prokaryotic mRNAs, or fragmented samples from FFPE, it is possible to do rRNAdepletion

Fragmentation of template to ‘fit’ sequencing platform

Fragmentation of RNA before RT – in alkaline solutions or enzymes

Fragmentation of cDNA – acoustic shearing, Dnase, transposons-based tagmentation method by Tn5: fragment cDNA and add adapter sequences at the same time

Tagmentation requires optimization of precise enzyme:DNAratio

Adapters and directionality

Naïve protocols - obtain reads from cDNA fragment. BUT the link with the sense or antisense strand is broken.

Stranded protocols - generate reads from one strand, corresponding to the sense or antisense strand (depending on the protocol)

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

Adding adapters directly to the 5’ or 3’ of the RNA

Incorporating dUTP in the second strand of cDNA

How to extract my RNA?

Step 1: Sample collection and protection

Step 2: RNA preparation

Step 3: QC of isolated RNA

Step 4: Storage of isolated RNA

RNA extraction method needs to be calibrated per project!

Step 1: Sample collection and protection

Lytic agent or denaturant must be in contact with cellular contents when cells are disrupted – problematic if:

Tissues or cells are hard (bones, roots)

Contain capsules or walls (yeast, spores)

It is not possible to process sample immediately after collection - transport from another site, many samples in parallel

RNA stabilization solution, freezing in dry ice/liquid nitrogen

Step 2: RNA preparation

Origin of the sampleTissues high in nucleases, fatty tissues, samples with high amounts of inhibitors

The amount of sample that can be obtainedA cell line (millions of cells), rare FACS sorted population (few thousands of cells)

The amount of RNA requiredDepends on the method of choice for RNA-seq

RNA Extraction methods

• ‘Gold standard for RNA preparation• Sample is homogenized in a phenol-containing solution

(Trizol, Qiazol – for fatty tissues) and centrifuged• Sample is separated into there phases• The upper aqueous phase is recovered and RNA is collected

by alcohol precipitation and rehydration

Organic extractions

Organic Extractions

Benefits – Rapid denaturation of nucleases and stabilization of RNA Scalable format Cheap, simpleDrawbacks – The use and associated waste of organic reagents Laborious and manually intensive processing Difficult to automate Requires a large amount of input sample

Filter-based, Spin Basket format

• Utilize membranes seated at the bottom of a small plastic basket

• Samples are lysed in a buffer that contains RNAse inhibitors (usually guanidine salts), and nucleic acids are bound to the membrane by passing the lysate through the membrane using centrifugal force

• Samples are than washed and eluted • Hybrid methods combine organic extraction with

purification by spin basket

Filter-based, Spin Basket format

Benefits – Convenient and easy to use Scalable format, ability to automateDrawbacks – Propensity to clog with particulate material Retention of large nucleic acids such as gDNA Fixed binding capacity Carryover of salts when using a sub-optimal sample input

Magnetic particle methods

• Small (0.5-1um) magnetic particles that bind DNA/RNA• Samples are lysed in a buffer that contains RNAse inhibitors

and allowed to bind to the magnetic particle• Following magnetization, samples are washed and RNA is

eluted of the magnetic particles

Magnetic particle methods

Benefits – No risk of filter clogging Solution-based binding kinetics increase target capture Rapid, easy to use, ability to automateDrawbacks – Potential carry-through of magnetic particles in the eluted

sample Slow migration of magnetic particles in viscous solution Capture/release of particles can be laborious

Direct lysis methods

• Perform sample preparation, not purification• Utilizes lysis buffer formulations that disrupt samples,

stabilize nucleic acids, and are compatible with downstream analysis

• A sample is mixed with lysis agent, incubated under specific conditions and used directly for downstream analysis

• By eliminating the need to bind and elute from solid surfaces, it avoids bias and recovery efficiency effects

Direct lysis methods

Benefits – Fast and easy Highest potential for accurate RNA representation Can work well with very small samples Scalable, possible to automateDrawbacks – Impossible to perform traditional analytical methods for

RNA yield Dilution based (most useful) Potential for sub-optimal performance, requires

optimization for downstream processes

Step 3: QC of isolated RNA

RNA quantityNanodropQubitReal Time PCR

RNA quality and purityNanodrop

RNA integrityElectrophoresis gel, Bioanalyzer, TapeStation

Nanodrop – for RNA quantity and purity

• Measures UV absorption

• RNA has a maximum absorption at 260 nm

• UV absorbance depends on pH of RNA solution

A260/280 – level of protein contamination Pure RNA =2.1Acceptable: 1.8-2.0

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

Qubit – measures low input samples

• Fluorometer, provides an accurate and selective method for the quantitation of low-abundance RNA samples.

• Can measure RNA or DNA depending on the kit used

• High sensitivity

RNA- integrity Gel electrophoresis

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

mRNArRNA

rRNA

Higher molecular weight bands – can indicated DNA contamination

Smearing below rRNA indicates poor RNA quality

RNA integrity – Bioanalyzer / Tapestation

A miniaturized version of agarose and acrylamide gels

RIN – RNA Integrity Number – quality score for total RNA

10 – maximum RNA integrity

Step 4: Storage of isolated RNA

RNase free environment Storage in -80oC Small aliquots – avoid freeze thaw cycles RNA storage solution (10mMTris-HCl, pH7.5)

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 (sortedcells, 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 atdifferent 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 P7Read1 Read2

Insert to sequence

Read to align to genome

Read 2 to obtain cell and molecule barcodes

Mars-seq Workshop in the Sandbox

• 3 days hands-on workshop

• Standard RNA material

• 1 representative per lab

Thank you for listening

Happy Sequencing