introduction to next-generation sequencingthaiviro.org/upload/์ngs-pdf/9 อ....

Introduction toNext-Generation Sequencing

Associate Prof. Sunchai Payungporn, Ph.D.

Department of Biochemistry

Faculty of Medicine

Chulalongkorn University

E-mail: [email protected]

Next-Generation Sequencing (NGS)

Highly parallel DNA sequencing technologies that produce millions of reads for a low cost and in a short time

http://upload.wikimedia.org/wikipedia/commons/2/2e/Mapping_Reads.png

First generation sequencing VS Next-generation sequencing

http://cmr.asm.org/content/29/4/837/F5.expansion.html

Next-generation sequencing platforms based on company

https://www.youtube.com/watch?v=fCd6B5HRaZ8

https://www.youtube.com/watch?v=zjEQPGDx-J4

https://www.youtube.com/watch?v=DyijNS0LWBY

https://www.youtube.com/watch?v=v8p4ph2MAvI https://www.youtube.com/watch?v=E9-Rm5AoZGw




https://www.youtube.com/watch?v=v8p4ph2MAvI

https://www.youtube.com/watch?v=E9-Rm5AoZGw

http://en.annoroad.com/news/company/51.html



http://www.bgi-agro.com/en/technology/41



http://www.medsantek.com.tr/index_en.php

Ion Torrent-PGM Ion Torrent-Proton IIIon S5 / S5 XL System



https://www.pacb.com/applications/whole-genome-sequencing/human/



https://mms.businesswire.com/media/20180319006158/en/646842/5/nanopore-product-family.jpg?download=1



Comparisons among different NGS platforms

https://twitter.com/albertvilella/status/946101154005639170

Comparisons among different NGS platforms

Summary of different NGS platforms

Platform Advantages Disadvantages

Illumina High data outputHigh accuracy

Short reads

BGI-SEQ High data outputLow cost/run

Short reads

Ion torrent Very fast run Error at repeated bases

Pacific Biosciences Very long reads High error rates

Oxford Nanopore Very long readsPortable

Quite high error rates

NGS terms: types of sequencing

Terms Description

Amplicon Sequencing High-throughput sequencing of DNA fragments obtained by conventional PCR.

De Novo Sequencing Sequencing of genetic material with no reference sequence available.

Re-Sequencing Sequencing of genetic material with reference sequence available.

Exome Sequencing Sequencing parts of genome made of exons.

RNA-Seq Sequencing of total RNA.

Chip-Seq For detecting transcription factor binding sites and histone modifications

NGS terms: sample processing

Terms Description

Sample Enrichment Preparation of a sample so that it contains the maximum amount of the

genetic material in question.

Fragmentation Splitting of genetic material into fragments of desired sizes: mechanically

(nebulisation, sonication) or enzymatically

Library A set of nucleic acid fragments which has undergone all processing steps and

is ready for actual sequencing.

Multiplex A library containing various samples labelled with barcodes.

Barcode or index A short unique sequence through which you can identify different samples

pooled into a single library.

Library preparation

Whole genome sequencing

Target ampliconssequencing

RNAsequencing

Size selection

& Adaptor ligation

From: ”Application of next-generation sequencing technologies in virology”Journal of General Virology (2012), 93, 1853–1868

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Multiplexing and barcoding

https://www.illumina.com/content/dam/illumina-marketing/images/technology/multiplexing-overview-figure.gif

NGS terms: methods of reading

Terms Description

Single-End Reads A method of reading a fragment where the fragment is read from one end only during

sequencing.

Paired-End Read A method of reading a fragment where the fragment is first read from one end and then

from the other.

Mate Pair-End Read Strategy for sample preparation where the longer fragment (thousands of bases) is

circularized using labelled adapters, the molecule is subsequently fragmented, but only

the fragments containing the labelled adapters are sequenced.

Paired-End VS Mate Pair-End

Paired-End + Mate Pair-End

NGS primary analysis

FASTQ files

NGS terms: primary analysis results

Terms Description

Output Capacity A number of read bases in sequencing, typically measured in thousands to

trillions of bases (kb, Mb, Gb, Tb), can be related to an experiment

Read Data output from the analysis of a single fragment (sequence).

Read Accuracy Indicates the occurrence of errors (in %) after primary analysis.

Read Length The number of read bases per fragment, respectively the maximum length of

the fragment, which can be sequenced at a time (indicated in bases).

Secondary analysis: FASTQ format@ followed by description

(Sequence in FASTA format)

(ASCII character)

Phred quality score (Q) = ASCII value - 33

(Q30)

@ Instrument: Run ID: Flowcell ID: Lane: Tile: X:Y ReadNum:FilterFlag:0:SampleNumber

ASCII: American Standard Code for Information Interchange

NGS secondary analysis

FASTQ files

Quality trimming

Adaptors trimming

Analysis specified by applications

• Map reads to reference sequences (SAM, BAM file)

• Alignment variant calling (VCF File)

• De novo assembly (Fragment Contig Scaffold)

• Read identification taxonomic classification (OTUs)

• Count reads expression levels / relative abundance

Trim bases with Q<30Trim adaptor sequences

• Read Depth: DNA = number of times a nucleotide is read

RNA = total number of reads per sample

• Coverage: This value indicates the coverage of an analysedsequence with respect to its length, usually expressed as a percentage;sometimes the term is also used for the depth of reading.

NGS terms: depth and coverage

Average read depth = 2.80

NGS terms: Map reads to reference sequences

Terms Description

SAM File File containing alignment of fragments together with quality indicators and possibly other

information.

BAM File Binary version of SAM file, a typical output of the secondary phase of data analysis.

VCF File A file containing information about the sequence variants identified, a typical output of

the secondary phase of data analysis.

Variant Calling Process of detection of sequence variants in the sequences obtained.

SNP Calling Process of detecting SNPs in the sequences obtained.

SNP Single-Nucleotide Polymorphism = sequence divergence in the range of a single base.

InDel Insertion/deletion = sequencing divergences that can cause a reading frame shift.

NGS terms: de novo assemblyTerms Description

Assembly Assembly of fragment sequences into higher order structures based on their overlap

and reference sequence, where appropriate.

Fragment A short stretch of nucleic acid resulting from the fragmentation of longer stretches and

sequenced. The required size of a fragment is specific to the type of experiment and

sequencer possibilities.

Contig The first level of the association of fragment sequences to higher structures (Fragment -

> Contig).

Scaffold The second level of the association of fragment sequences to higher structures

(Fragment -> Contig -> Scaffold).

Applications of NGS in virology• Viral genome characterization

de novo sequencing (new emerging viruses)

Resequencing (minor mutations analysis)

• Deep sequencing of target gene

Antigenic variations

Drug resistant mutants

High resolution genotyping

• Transcriptome (virus-host interaction)

Alternative splicing

RNA editing

Differential gene expression profiling

MicroRNA expression profiling

• Metagenome

Virome

Pathogen identification

From: “The next-generation sequencing technology and application” Protein Cell 2010, 1(6): 520–536

introduction to next-generation sequencingthaiviro.org/upload/์ngs-pdf/9 อ....

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