SnapShot High–Throughput Sequencing Applications

A

A

A

A

A

A

A

A

A

A

A

A

60S

A G O

40S

60S60

S

A A A A A A A A

A A

40S

40S

Pol II

TF TF

G e

n o

m

i c

D N

A

TF

TF

T F

TF

TF

R

B

P

R B P

R

B

P

RBP

RBP

RBP

Small RNA Sequencing

e.g., microRNA/Piwi-interacting RNA pro?ling

Argonaute (Ago) HITS-CLIP

Mapping interactions between microRNAs and mRNAs 5′ UTR3′ UTR

Start

codon

Stop

codon

CDS

CDS

Ribosome Pro?ling

Sequencing ribosome-protected mRNA fragments

Mapping ribosome footprints within

transcripts at nucleotide level resolution mRNA

DEGRADATION TRANSLATION

SUPPRESSION

RNA-induced silencing complex

(RISC)

microRNA

ACTIVE TRANSLATION

Ribosome

Protein

mRNA EXPORT

Constitutively

spliced exon

Alternatively

spliced exon

Nascent RNA

SPLICING

TRANSCRIPTION

RNA-binding

proteins

CpG

METHYLATION

Nucleosome

Cap

C G

T

A

C H

3

H3K36me3

Chromosome

SNP

Polymerase II

complex

Transcription

factors

Polymerase II

Intron

Poly(A) tail

Crosslinking Immunoprecipitation Sequencing

(CLIP-Seq/HITS-CLIP)

Transcriptome-wide RNA-binding protein (RBP) maps

Modi?ed Clip For Site-speci?c Crosslinking

Photoactivatable ribonucleoside-enhanced CLIP (PAR-CLIP)

Individual nucleotide resolution CLIP (iCLIP)

Eg. Nova RNA-binding regulatory map

Chromatin Immunoprecipitation Sequencing

(ChIP-Seq)

Nucleosome component, Transcription factor (TF),

RNA polymerase II (Pol II) occupancy

Histone methylation or acetylation

Methyl-Seq/Bisul?te-Seq (DNA methylation status)

DNase-Seq (DNase hypersensitivity)

R

e

l

a

t

i

v

e

e

n

r

i

c

h

m

e

n

t

5′ UTR3′ UTR

Intron

Exon Alt.

Exon

RNA Pol II/TF

H3K36me3

H2K27ac

Transcriptome Sequencing/RNA-Seq

Total RNA, total RNA minus rRNA, poly(A)-selected RNA

Gene expression pro?ling

Long noncoding RNA pro?ling

Alternative splicing and trans-splicing pro?ling

Alternative polyadenylation pro?ling

Mapping transcription initiation sites

Mapping RNA editing sites (coupled with DNA-Seq)

Targeted RNA-Seq, Direct RNA-Seq, Strand-speci?c

RNA-Seq, Nascent RNA-Seq

Reads mapped to exons Reads mapped to junctions

Genome Sequencing/Resequencing

Genome-wide polymorphism and mutation mapping

Genome assembly

5′ UTR

1Banting and Best Department of Medical Research, University of Toronto, Toronto, ON M5S 3E1, Canada

1Banting and Best Department of Medical Research, University of Toronto, Toronto, ON M5S 3E1, Canada Table 1. High-Throughput Next Generation Sequencing (NGS) Platforms

Platforms Amplification; Sequencing

Chemistries

Detection

Read length a

SE-single end; PE-paired end)

Reads per lane/No. of

lanes (X2 for dual flow

cell)

Run time b

Illumina HiSeq 2000

https://www.sodocs.net/doc/2910817917.html,

Bridge Amplification; Synthesis Fluorescence100 bp (PE)50 × 106 / 8 (X2)11 days

Roche/454’s GS FLX Titanium

https://www.sodocs.net/doc/2910817917.html,

Emulsion PCR; Synthesis Luminescence400 bp (SE) 1 × 10610 hr

Life/APG’s SOLiD 3

https://www.sodocs.net/doc/2910817917.html,

Emulsion PCR; Ligation Fluorescence50 bp (PE)40 × 106 / 8 (X2)14 days

Polonator G.007

https://www.sodocs.net/doc/2910817917.html,

Emulsion PCR; Ligation Fluorescence13 bp (PE)10 × 106 / 8 (X2) 4 days

Helicos BioSciences HeliScope

https://www.sodocs.net/doc/2910817917.html,

No amplification; Synthesis Fluorescence35 bp (SE)20 × 106 / 25 (X2)8 days

Pacific Biosciences

https://www.sodocs.net/doc/2910817917.html,

No amplification; Synthesis Fluorescence>1000 bp (SE)150,000 per SMRT cell N/A

Ion Torrent

https://www.sodocs.net/doc/2910817917.html,

Emulsion PCR; Synthesis Change in pH200 bp (SE)Variable<2 hr

Complete Genomics Analysis Platform https://www.sodocs.net/doc/2910817917.html, DNA nanoballs; Ligation Fluorescence

Complete genomic analysis service at 40× human genome coverage;

>90% of the full genome resolved (both alleles)

400 human genomes

per month

Modified and updated from Metzker (2010).

a Average read length.

b Run time for full sequencing experiment.

Sequencing technologies in development: Nanopore sequencing (Oxford Nanopore: https://www.sodocs.net/doc/2910817917.html,; Nabsys: https://www.sodocs.net/doc/2910817917.html,). Electron Microscopy base sequencing (Halcyon Molecular: https://www.sodocs.net/doc/2910817917.html,; ZS Genetics: https://www.sodocs.net/doc/2910817917.html,).

High-throughput, next-generation sequencing (NGS) technologies have revolutionized genomics, epigenomics and transcriptomics studies by allowing massively parallel sequencing at a relatively low cost. In this SnapShot, we highlight the increasingly diverse applications of NGS, including genome sequencing/resequencing, transcriptome sequencing, small RNA sequencing, analysis of DNA/RNA-protein interactions, and ribosome profiling. In addition, we provide a quick guide (Table 1) to the currently available NGS platforms, together with their underlying methodologies and unique features.

Genome Sequencing/Resequencing

Whole-genome sequencing/resequencing and targeted genome resequencing have been used extensively for sequence polymorphism discovery and mutation mapping. These applications are rapidly advancing our understanding of human health and disease and are also facilitating the de novo assembly of uncharacterized genomes.

DNA-Protein Interactions and Epigenome Sequencing

Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) is a powerful technique for genome-wide profiling of DNA-protein interactions and epi-genetic marks. It has facilitated a wide range of biological studies, including transcription factor binding, RNA polymerase occupancy, nucleosome positioning and histone modifications. Complementary methods being used to study chromatin structure and composition are Methyl-Seq and DNase-Seq for profiling DNA methylation and DNase-hypersensitive sites, respectively.

Transcriptome Sequencing/RNA-Seq

The introduction of transcriptome sequencing/RNA-Seq has provided a new approach for characterizing and quantifying transcripts. In general, total RNA, rRNA-depleted total RNA, or poly(A)-selected RNA are converted to double-stranded cDNA fragments that are then subjected to high-throughput sequencing. This strategy has been applied for profiling mRNA and noncoding RNA expression, alternative splicing, trans-splicing, and alternative polyadenylation and for mapping transcription initiation, termination, and RNA editing sites. Related applications include targeted RNA-Seq, direct RNA-Seq, strand-specific RNA-Seq, and nascent RNA-Seq (e.g., global run-on sequencing, GRO-Seq, and native elongating transcript sequencing, “NET-Seq”).

RNA-Protein Interactions

CLIP-Seq, also known as HITS-CLIP, is a method employing in vivo crosslinking of RNA to protein followed by immunoprecipitation and high-throughput RNA sequencing to generate transcriptome-wide RNA-protein interaction maps. Modified CLIP-Seq technologies, such as PAR-CLIP (photoactivatable ribonucleoside-enhanced CLIP) and iCLIP (individual nucleotide resolution CLIP), have been applied to increase crosslinking efficiency and resolution.

Small RNA Sequencing

Similar to RNA-Seq, sequencing of size-selected short RNA provides insight into small RNA populations in different organisms, tissue and cell types, developmental stages, and disease states. It has greatly contributed to our understanding of the functions and regulatory mechanisms of different classes of small RNAs, such as microRNAs (miRNAs) and Piwi-interacting RNAs (piwiRNAs). With the recent development of Argonaute (Ago) HITS-CLIP, it is possible to simultaneously detect Ago-bound microRNAs and mRNA segments, which enables the large-scale mapping of in vivo miRNA-mRNA interactions.

Ribosome Profiling

In addition to the profound impact of NGS on transcriptomic studies, the development of methods enabling high-throughput sequencing of ribosome-protected mRNA fragments has provided a powerful tool for the analysis of translationally engaged mRNA on a genome-wide scale.

Additional Applications and Future Directions

High-throughput sequencing is a rapidly evolving technology and will likely continue to change the face of “omics” studies in the years to come. Although NGS technologies power a wide spectrum of current research applications, new innovations are continually being developed. These include barcode sequencing strategies for multiplexing the analysis of samples, metagenomic analyses, protein-protein interactome mapping (“Stitch-Seq”), and high-definition measurement of DNA-affinity landscapes (HiTS-FLIP). Future technical advances and applications are expected to further revolutionize our understanding of evolutionary biology and genotype-phenotype relationships and ultimately to bring personalized medicine into the clinic.

1Banting and Best Department of Medical Research, University of Toronto, Toronto, ON M5S 3E1, Canada

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