biotech autumn2012-02-ngs2
TRANSCRIPT
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