NGS

part 2

NGS workflow

Создание библиотеки

Подготовка субстрата Секвенирование

Анализ данных

NGS workflow

Конструирование библиотек

ДНК для приготовления библиотек должна быть фрагментирована

- Рестрикция – «коктейль» рестриктаз

- УЗ-фрагментация

- Без фрагментации

Ion Torrent library workflow

DNA

Shear DNA

Fragmented DNA End Polished Fragments

Adapter Ligation, Nick-repair

& Size Selection

PCR Amplify Template

Preparation

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Illumina library

Ion Torrent vs. Illumina library workflow

Nextera DNA sample preparation kit (рекомбиназный способ)

Quantification Method Sensitivity

E-Gel® System 5 ng / μL

Bioanalyzer TM 2100 DNA 1000 chip

DNA HS chip

0.1 - 50 ng / μL (50 bp - 7 kb)

Qubit® Fluorometer

10 pg /μL - 100 ng /μL

qPCR 0.005 ng /μL

Library QC - Evaluate library quantity/quality

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Size selection

E-Gele Pippin Prep

Bioanalyzer (Agilent)

Example of gDNA after shearing (good)

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Example: expected size distribution of fragmented genomic DNA

Example of gDNA after shearing (bad)

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Example: size distribution of oversized fragmented genomic DNA. Troubleshoot: ie fragment for longer time.

Example of good DNA Library (end of library prep)

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Library

Example: BioanalyzerTM analysis of good final fragment DNA library (for 100 bp reads). Notice library

profile is single, narrow peak.

Example of bad DNA Library – with primer dimers

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Example: library with primer-dimer contamination, resulting in inefficient Template Prep. Re-purify library with AMPure® beads.

Unwanted primer-dimers

Library

Example of bad DNA Library – with concatemers

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Example: an over-amplified library with concatemer products, resulting in inefficient Template Prep. Re-purify with size-selection and then re-quantify.

Unwanted concatemers

Library

Fragment Shear Pippin Size Selection

___ ___

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Shared DNA, Library

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library insert fwd primer

rev primer

probe

qPCR (real-time PCR)

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Example qPCR data

• E. Coli DH10B Control Library used as Standards in Red (triplicates)

• Ion Libraries (triplicates)

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Example qPCR data

Optimizing the library input concentration

Amplification

Low DNA High DNA

Increasing [DNA]

“Mixed” read No read

Optimal DNA

Too little library input can result in insufficient positive,

or “live”, spheres for sequencing

Too much library input to template prep can result in

too many “mixed” reads

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Microbial sequencing

Mitochondrial sequencing

Amplicon sequencing • Multiplexed amplicon sequencing for rapid detection of germline and somatic mutations

Targeted resequencing by target enrichment

RNA-Seq • Whole-transcriptome human RNA • Small RNA

Chip-Seq

Copy number detection

Applications

Typical RNA-seq experiment

Total RNA sequencing

Ligase Enhanced Genome Detection (LEGenD™) technology

mRNA-Seq library preparation (ревертазный способ)

Small RNA-Seq library preparation (РНК-лигазный способ)

Target sequencing

Таргетное секвенирование - Целевое секвенирование определенных интересующих участков

генома с предварительной наработкой фрагментов и созданием библиотеки – Target enrichment

Преимущества:

• Увеличение покрытия без увеличения стоимости секвенирования.

• Полученные данные гораздо проще обрабатывать и хранить.

Target enrichment

• PCR

– short amplicons

– long amlicons

• Hybridization

– solution phase hybridization

– solid phase hybridization

Ion PGM Library preparation

Ion PGM Library preparation

Long amplicons

Методы пробоподготовки

• ПЦР

Ion AmpliSeq

•Up to 4,000 primers per pool

•One to hundreds of genes

•96 barcodes for multiplexing

TruSeq Amplicon (Illumina)

Fusion PCR

Fusion PCR

Microfluidic PCR

Hybrid capture

Solid Solution

Solution phase sequence capture using long RNA probes

SureSelect (Agilent) TruSeq (Illumina)

Solution phase sequence capture using molecular inversion probes (MIP)

MIP (molecular inversion probes)

HaloPlex Target (Agilent)

Solid phase sequence capture using DNA microarrays

NimbleGen (Roche)

Paired-End sequencing

Paired-End sequencing

Paired-End sequencing library

Detecting structural variants by paired-end mapping

Next generation sequencing based approaches to epigenomics

• Histone modification profiling (ChIP)

• DNA methylation profiling

– Enrichment based methods

– Bisulfite conversion based methods

– Methyl-sensitive restriction based methods

– Direct detection

ChiP-seq (chromatin immunoprecipitation sequencing)

Methylated DNA immuno-precipitation (MeDIP-Seq)

Bisulfite sequencing

Barcoding

Barcoding

Barcoding

Спасибо за внимание