NGS in Pathology Webinar - Agilent€¦ · Webinar Introduction to RNA Seq Jennifer Carter ......

NGS in Pathology Webinar

Introduction to RNA Seq

Jennifer Carter Jones, PhD

May 10 2016

For Research Use Only. Not for use in diagnostic

procedures.

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Topics for today’s presentation

Significance of RNA Profiling

Examples of RNA-Seq utility Disease Research

Introduction to RNA Seq workflow

Challenges with samples and sensitivity

Further information


procedures.

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The History of RNA Profiling and Sequencing


procedures.

1930 1940 1950 1960 1970 1980 1990 2000 2010 2012 year

2014 2016

Role of RNA

suspected

X-ray structure

of dsRNA Reverse

Transcription

discovered

Introns and

splicing

discovered

Pyrosequencing

developed Concept of

messenger

RNA

emerged and

Central

Dogma was

first

proposed

RNA

polymerase

purified

First whole

nucleic acid

sequenced

(tRNA)

Sanger

Sequencing

developed

qPCR and

qRT-PCR

introduced

Dideoxy

sequencing

2nd Generation

Sequencer

developed

(454) “ION”

sequencer

released

First RNA-Seq

publication

PacBio

SMRT

Sequencing

(3rd

Generation)

Oxford

Nanopore

Sequencing

TCGA portal

created

Expression

Atlas Database

Automated

capillary

sequencing

(ABI)

Single cell

RNA-Seq

published

Circulating

tumor Cell

RNA-Seq

mRNA and

miRNA

found in

exosomes

Agilent

introduces

RNA

Enrichment

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procedures.

Central Dogma of Molecular Biology

DNA RNA Protein

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procedures.

Central Dogma of Molecular Biology Complexity

DNA RNA Protein

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Many Technologies for Transcriptome Studies


procedures.

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What is RNA-Seq?

Application of massively parallel next generation sequencing to

detect and quantify the RNA from a sample


procedures.

seqwright.com

Isolate RNA from

samples

Prepare library

Obtain sequences;

Analyze data

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What is RNA-Seq?

RNA-Seq, also referred to as Whole Transcriptome Sequencing, is the use

of next generation sequencing (NGS) to detect and quantitate the RNA

molecules within a given cell or cells.


procedures.

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What is RNA-Seq?




• Tissue and temporally regulated, reflects the molecular activity within

the cell at that moment in time.


procedures.

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What is RNA-Seq?






• Reflects regulatory activity within the cell that is not discernable from

the genomic sequence alone.


procedures.

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What is RNA-Seq?








• Can identify differentially expressed genes, isoforms, expressed

genetics variants from specific alleles as well as gene fusions, novel

isoforms and splicing variants.


procedures.

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What is RNA-Seq?








• Can identify differentially expressed genes, isoforms, expressed

genetics variants from specific alleles as well as gene fusions, novel

isoforms and splicing variants.

• Reflects the complexity of the molecular state of individual cells within

tissue.


procedures.

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Functional Data generated from RNA-Seq


procedures.

• Differential expression

analysis

• Development of a gene

signature

• SNP detection

Profiling

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procedures.


analysis


signature

• SNP detection

Profiling

Gene Expr.

Alt. Splicing

SNP

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• Novel transcript discovery

• Novel gene fusions

• Novel exons/alt. start sites

• Alternative splicing


procedures.


analysis


signature

• SNP detection

Profiling Discovery

Gene Expr.

Alt. Splicing

SNP

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Further information


procedures.

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Combined DNA and RNA Sequencing: Genomic correlates of response to CTLA-4 blockade in metastatic melanoma Van Allen EM, Miao D, Schilling B, Shukla SA, Blank C, Zimmer L, Sucker A, Hillen U, Geukes Foppen MH, Goldinger SM, Utikal J, Hassel JC, Weide B, Kaehler KC, Loquai C, Mohr P, Gutzmer R, Dummer R, Gabriel S, Wu CJ, Schadendorf D, Garraway LA. Science. 2015 Oct 9;350(6257):207-11. doi: 10.1126/science.aad0095. Epub 2015


procedures.

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• Whole exomes from pretreatment melanoma tumor biopsies and

matching germline tissue samples. Transcriptome analysis for a

subset of these was used to analyze for tumor microenvironment

signatures associated with clinical benefit of ipilimumab treatment.

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procedures.

• Findings suggest that expression of immune checkpoint

molecules themselves may correlate with immunotherapy

response

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procedures.

• Integrating exome and transcriptome sequencing data help

inform the relative contributions of tumor immunogenicity and

host immune infiltration in determining clinical benefit of

treatment.

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Targeted RNA Sequencing: STK10 missense mutations associated with anti-apoptotic function. Fukumura K1, Yamashita Y, Kawazu M, Sai E, Fujiwara S, Nakamura N, Takeuchi K, Ando M, Miyazono K, Ueno T, Ozawa K, Mano H. Oncol Rep. 2013 Oct;30(4):1542-8. doi: 10.3892/or.2013.2605. Epub 2013 Jul 9..


procedures.

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procedures.

• Targeted RNA-Seq was performed with peripheral T-cell

Lymphoma specimens to simultaneously detect point

mutations/indels/gene fusions in a single experiment.

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procedures.

• Discovered a STK10 amino acid substitution recently deposited

as a single nucleotide polymorphism (SNP) in the 1000 genome

database that turned out to exert anti-apoptotic effects.

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procedures.

• Results suggest that STK10 may contribute to carcinogenesis,

either through polymorphism or somatic mutations, by

suppressing apoptotic signaling in cancer

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General examples


procedures.

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General examples


procedures.

• Multigene mRNA signatures used in clinical management of disease.

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General examples


procedures.


• Detection of known and unknown oncogenic Gene fusions (AML with

RNX1-RUNX1T1) and alternative splicing (EGFRvIII).

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General examples


procedures.




• Signatures of immune-related disease risk and disease-related immune

response.

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General examples


procedures.





response.

• Detection allele-specific expression leading to increased disease risk

(due to imprinting, abnormal methylation or nonsense-mediated decay)

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General examples


procedures.





response.



• Non-invasive examining of extracellular RNA (exRNA) for signatures of

disease (detection/disease monitoring, as well as course of treatment)

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General examples


procedures.





response.



• Non-invasive examining of extracellular RNA (exRNA) for signatures of

disease (detection/disease monitoring, as well as course of treatment)

• Detection and identification of relevant RNA virus in infectious disease

(direct influenza virus detection and Ebola outbreak tracking)

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Further information


procedures.

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How DNA-Seq and RNA-Seq Differ

DNA-Seq RNA-Seq


procedures.

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DNA-Seq RNA-Seq


procedures.

How DNA-Seq and RNA-Seq Differ

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Basic RNA-Seq Library Prep Steps


procedures.

Isolate Total RNA and QC

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procedures.

Isolate Total RNA and QC PolyA selection or

ribosomal and/or globin

depletion*

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procedures.



depletion*

Chemical or enzymatic

fragmentation

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procedures.



depletion*

1st strand cDNA synthesis Chemical or enzymatic

fragmentation

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procedures.



depletion*

1st strand cDNA synthesis

2nd strand cDNA synthesis


fragmentation

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procedures.



depletion*




fragmentation

Repair ends & adaptor

ligation

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procedures.



depletion*




fragmentation


ligation

PCR Amplification and

optional barcoding

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procedures.



depletion*



Sequence


fragmentation


ligation

PCR Amplification and

optional barcoding

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Why Strand Specific RNA Sequencing?

• To discover antisense transcripts with potential regulatory roles

• To identify overlapping transcripts

• To accurately measure the expression level of a given transcript


procedures.

5’ 3’

3’ 5’

5’ 3’

3’ 5’

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Why Strand Specific RNA Sequencing?

• To discover antisense transcripts with potential regulatory roles

• To identify overlapping transcripts

• To accurately measure the expression level of a given transcript


procedures.

Sultan et al, 2012 Biochem Biophys Res Commun 15;422(4):643-6

5’ 3’

3’ 5’

5’ 3’

3’ 5’

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Indexing (barcoding): More Samples/Lane Used with Target-Enriched Libraries

• Index is a nucleotide barcode, usually 6-8 bp in length

• Links reads to samples

• Allows multiple samples to be mixed on single lane to take full

advantage of sequencing capacity

• Also known as multiplexing


procedures.

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Quality Metrics of RNA-Seq Libraries


procedures.

Levin et al. (2010), Nature Methods 7(9):709-15

• Library Complexity

• Coverage (reads/locus)

(10X, 20X, 100x)

• Sequencing Depth

(number of reads/sample)

• Mapping Efficiency (how

many reads can be

aligned to

genome/transcriptome)

• Strand-Specificity

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Analysis of RNA-Seq Libraries


procedures.

1. Aligning reads for transcript identification and counting

Reads (paired–end)

Mapped to Genome*

Transcript

identification

and counting

Transcript

discovery

and

counting

Use of a

“gapped”

mapper

TopHat

STAR

Reads (paired–end)

Mapped to

Transcriptome

Transcript

identification

and counting

Use of a

“ungapped”

mapper

Bowtie

Cufflinks RSEM

Kallisto

transcript

Mapping to transcriptome

Mapping to genome

OR

Processed mRNA

Discovery Speed

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Analysis of RNA-Seq Libraries


procedures.

2. Determine Differential Expression

Transcript counts

Transcript Quantification

(RPKM, FPKM, TPM)

Significantly differentially

expressed genes within a

sample set.

Normalization/scaling RPKM = X raw # of reads

exon length 1,000,000

# of mapped reads in the sample

General analysis Correlation Analysis

Hierarchical Clustering

ANOVA

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Analysis of RNA-Seq Libraries Advanced Analysis


procedures.

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procedures.

3. Fusion, differential splicing and other RNA species

detection.

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procedures.


detection.

4. Visualization

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procedures.


detection.

4. Visualization

5. Integration with other sample data

Complexity

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Further information


procedures.

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procedures.

RNA sample source (quality and sensitivity to degradation)

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procedures.


• Formalin Fixed Paraffin Embedded sample RNA

- degraded/fragmented

- Crosslinked

- Low yield

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procedures.




- Crosslinked

- Low yield

• Circulating Tumor Cell/ single cell RNA

- Source cells difficult to isolate.

- Extremely low yield

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procedures.




- Crosslinked

- Low yield

• Circulating Tumor Cell/ single cell RNA

- Source cells difficult to isolate.

- Extremely low yield

• Exosomal and cell-free RNA


- Source cells/RNA difficult to isolate.

- Low yield

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Determining the Quality of RNA

HOW DO WE GET THERE with FFPE

RNA?

Calculate the DV200 % of fragments above 200nt.

Agilent currently suggest using samples with DV200 ≥ 20% with

the Direct to Capture Enrichment.


procedures.

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Determining the Quality of RNA


procedures.

HOW DO WE GET THERE with FFPE

RNA?

Calculate the DV200 % of fragments above 200nt.

Agilent currently suggest using samples with DV200 ≥ 20% with

the Direct to Capture Enrichment.

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Target Enrichment: It’s just like fishing…


procedures.

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procedures.

What is the basic concept?

• Isolate the transcripts of interest

• Transcripts that are captured/amplified from initial library (pre-capture library) undergo additional amplification and processing creating a post-capture library

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procedures.

Why perform target enrichment?

1. Sequence only your desired transcripts and minimize presence of high

abundance non target transcripts.

2. Sequence more samples per lane/run (i.e., multiplex)

3. Save time and money

4. Identify isoforms, fusions and variants in transcripts with increased reliability

and accuracy = Higher Coverage

What is the basic concept?

• Isolate the transcripts of interest

• Transcripts that are captured/amplified from initial library (pre-capture library) undergo additional amplification and processing creating a post-capture library

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New Direct to Capture SureSelect XT RNA Enrichment Library Prep


procedures.

100 (high quality) - 200 ng (FFPE) purified

total RNA (DV200 ≥ 20)

Capture-ready cDNA

libraries

@ 6 hours

(@ 2.5 hours hands on)

1 Speed-vac RNA and resuspend

in Fragmentation buffer.

Priming & denaturation (10min)

2

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procedures.



Capture-ready cDNA

libraries

@ 6 hours


1

1st strand synthesis w/

actinomycin D (60min)

3 4

Synthesize first strand

SPRI cleanup

(30min)

Speed-vac RNA and resuspend



2

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procedures.



Capture-ready cDNA

libraries

@ 6 hours


1



3 4


SPRI cleanup

(30min)




2

5 6 2nd strand synthesis (dUTP)

5’ Phosphorylation & end-

repair (60min)

Synthesize second strand

SPRI cleanup

(30min)

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procedures.



Capture-ready cDNA

libraries

@ 6 hours


1



3 4


SPRI cleanup

(30min)




2

5 6 2nd strand synthesis (dUTP)

5’ Phosphorylation & end-

repair (60min)

Synthesize second strand

SPRI cleanup

(30min)

UDG treatment

& PCR (60min)

Prepare libraries (200ng) for Capture (SureSelect XT Protocol*)

7 3’-dA

tailing

(30 min)

8 Adapter

Ligation

(15 min)

SPRI

cleanup

(30min)

9 10 11

SPRI cleanup

(30min)

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How SureSelect RNA Target Enrichment Works


procedures.

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Benefits and Limitations of Direct to Capture RNA-Seq


procedures.

Benefits Limitations

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procedures.

FFPE compatible (no

ribosomal depletion) and

lower input for non-FFPE

1


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procedures.

FFPE compatible (no



1

Less bias/more focus on

coding sequence

2


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procedures.

FFPE compatible (no



1


coding sequence

2

Increased sensitivity to

splicing variants over RNA-Seq

3


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procedures.

FFPE compatible (no



1


coding sequence

2



3

Benefits

Increased sensitivity

transcripts with lower

abundance

4

Limitations

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procedures.

FFPE compatible (no



1


coding sequence

2



3

Benefits



abundance

4

Requires capture library

(V6+UTR or custom)

1

Limitations

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procedures.

FFPE compatible (no



1


coding sequence

2



3

Benefits



abundance

4


(V6+UTR or custom)

1

Lower likelihood of detecting

completely novel transcripts

2

Limitations

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procedures.

FFPE compatible (no



1


coding sequence

2



3

Benefits



abundance

4


(V6+UTR or custom)

1



2

Not currently compatible with

FFPE RNA DV200 < 20

3

Limitations

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procedures.

FFPE compatible (no



1


coding sequence

2



3

Benefits



abundance

4


(V6+UTR or custom)

1



2

Not currently compatible with

FFPE RNA DV200 < 20

3

Decreased sensitivity on the

highest end of dynamic range

4

Limitations

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More Sequencing Means Greater Diversity of RNA Types


procedures.

Tarazona et al., Genome Res. 2011 Dec;21(12):2213-23

Need more reads to

detect transcripts with

lower abundance like

regulatory RNA than

for protein coding

genes

Protein Coding RNA

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How Much Sequencing is Needed? Depends on experimental goal


procedures.

Sequencing Depth

• Expressed as numbers of reads

per sample

• No set recommendation

30M-80M paired-end

reads/sample (Gene

Expression)

100-200M paired-end

reads/sample (alt splice,

novel transcripts,

fusions)

Illumina HiSeq 3000: 3 trillion

paired end reads/run(187 million

reads per lane); ~11 days

(2X100bp) high output dual flow

cells.

MiSeq: 24-30M paired end reads;

24 hours (2X150bp)

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RNA-Seq Target Enrichment in Cancer

Aim: RNA Target Enrichment of 467 Cancer Genes (~all Tyr Kinases + Genes from Cancer Gene

Census); Overall >800 target transcripts

Method: Enrich cDNA from K-562 CML cell line cDNA libraries and compare results from before

and after target enrichment

No bias


procedures.

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Benefits of RNA-Seq Target Enrichment in Cancer

Whole transcriptome analysis requires >40x more sequencing to

achieve same depth for targeted regions


procedures.

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Which technology is best suited for your project?


procedures.

RNA-Seq

• Differential expression as well novel transcripts, fusions, splice variants

• Higher cost.

• Open platform but lower sensitivity

RNA Target Enrichment

• Differential expression studies, splice variants and fusions

• Lower cost with higher sample throughput

• Pre-determined content but more sensitive

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procedures.

Profiling? Discovery?

RNA-Seq

• Differential expression as well novel transcripts, fusions, splice variants

• Higher cost.






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procedures.

Profiling? Discovery?

What scientific question am I trying to address?

How much sample material do I have?

How much money do I have?

How many replicates will I need?

RNA-Seq

• Differential expression, splice variants, fusions, as well novel transcripts

• Higher cost.






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Further information


procedures.

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Key Considerations for RNA-Seq

• Choose the approach most suited for your research question (whole transcriptome or targeted enrichment)

• Think outside “the box”: sequencing costs + library prep costs + reagents + time + analysis

• Consult your friendly statistician/bioinformatician to determine how many total reads/sample, how many reads per locus, and how many (biological) replicates are needed to address the biological question

• Develop a plan for data analysis; practice with NGS data deposited in GEO, SRA, Encode


procedures.

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Why we know NGS & more… Agilent Technologies Solutions for the NGS workflow


procedures.

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Why we know NGS & more… Agilent Technologies Solutions for the NGS workflow

The Gold Standard for Sample QC

2100 Bioanalyzer Instrument & Kits

2200 TapeStation Instrument & Kits


procedures.

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Why we know NGS 101 & more… Agilent Technologies Solutions for the NGS workflow




The Leader in NGS Target Enrichment

SureDesign

SureSelect

HaloPlex

Bravo Automation


procedures.

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NGS Analysis Software

GeneSpring

- Strand-NGS

Globus/Navipoint


SureDesign

SureSelect

HaloPlex

Bravo Automation


procedures.

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NGS Analysis Software

GeneSpring

- Strand-NGS

Globus/Navipoint

Validation Technologies

qPCR- Aria system & Brilliant reagents

Microarrays- Gene Expression & miRNA


SureDesign

SureSelect

HaloPlex

Bravo Automation


procedures.

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Contact Us

800.227.9770

[email protected]

www.agilent.com/genomics


procedures.

http://www.agilent.com/genomics

NGS in Pathology Webinar - Agilent€¦ · Webinar Introduction to RNA Seq Jennifer Carter ......

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