how to cluster and sequence an ngs library (james hadfield160416)

‘How to prepare, cluster and sequence an NGS library’

AN OVERVIEW OF NGS IN THE GENOMICS CORE– Introduction– Understanding library prep– Understanding clustering and sequencing– Understanding instruments– NGS QC– NGS applications

A potted history of Illumina sequencing

Understanding library prep

Adapter ligation

End-repairAdenylation

BioAnalyserqPCRPCR

“The next ten slides are the most important I’ll show today “

Understanding library prep

Understanding library prep

Understanding library prep

Understanding library prep

Understanding library prep

Understanding library prep

Understanding library prep

Understanding library prep

Understanding library prep

Understanding library prep

– Text

Illumina adapters ask for Illumina letter!

ACACTCTTTCCCTACACGACGCTCTTCCGATCT

ADAPTER

PCR PRIMER

SEQ PRIMER

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT

CAAGCA

GAAGAC

GGCATA

CGAGCTCTTCCGATCT

Insert DNA

Index

PCR

Index SEQ

ACACTC

TTTCCC

TACACG

ACGCTCTTCCGATCT

InsertDNA A

||||||||||InsertDNAACTCGTATGCCGTCTTCTGCTTG P-GATCGGAAGAG

ACACTCTTTCCCTACACGACG CTCTTCCGATCT T

||||||||||

CTCGTATGCCGTCTTCTGCTTGP-GATCGGAAGAG

ACACTC

TTTCCC

TACACG

ACGCTCTTCCGATCT

T||||||||||

Oligonucleotide sequences © Illumina, Inc. All rights reserved.

Understanding library prep

“The next ten slides are the most important I’ll show today “

The library prep spike

[DN

A]

Illumina Processing

Understanding library prep – Nextera!

– Text

Understanding cluster generation (2500 etc)

Understanding cluster generation (2500 etc)

A) Diluted & denatured libraries are annealed to lawn oligos at their 3’ end, and a polymerase creates a covalently attached copy of the library molecule.B) The original strand is removed by denaturation with NaOH.C) In non-denaturing conditions the library molecule bends and hybridises to a lawn oligo complementary to the 5’ end, and a polymerase creates a second covalently attached molecule. This amplification is repeated to create a cluster with around 1000 copies of the original library molecule.

A B C

Understanding cluster generation (2500 etc)

D E C G H

D) Clusters are linearized by cleavage at the 3’ end of the original library molecule, and denaturation leaves the single stranded DNA which will be sequenced. A sequencing primer is hybridised* and sequencing-by-synthesis generates the first read in your fastq file.-) For single-end indexing the the SBS template is removed by denaturation, and the index 1 sequencing primer is hybridised ready to generate index1 (i7). Dual-indexing is complicated and differs on single- or paired-end flowcells but the process is essentially the same to generate index two (i5).E-G) For paired-end sequencing the SBS template is removed by denaturation, the cluster is re-amplified for several cycles, cleaved at the 5’ end the paired-end sequencing primer hybridised ready to generate read 2.

*Beware: if you create new adapters let us know if you need a custom sequencing primer

Understanding cluster generation (X Ten & 4000)Exclusion Amplification

The same hybridisation and solid-surface amplification occurs but in an all-in-one phase called “exclusion amplification” (ExAmp). Once a library molecule “lands” in a well it should occupy it completely.

Understanding cluster generation (X Ten & 4000)Exclusion Amplification

Understanding sequencing

Understanding sequencing: Sanger-seq

Understanding sequencing: Pyro-seq

Understanding sequencing: Sequencing-by-synthesis

Understanding “sequencing by synthesis”

Understanding “sequencing by synthesis”

Instrument “colours”

HiSeq, MiSeq 4-colour SBS

NextSeq 2-colour SBS

Firefly 1-colour SBS?

Instruments explained – HiSeq 2500 & 4000

Different sequencing configurations

2500 Rapid150M readsSE 50bp

85%Q30PE 250bp

75%Q30PE 150 2 days

2500 High output250M readsSE 50bp

85%Q30PE 125bp

80%Q30PE 125 6 days

4000 High output312M readsSE 50bp

85%Q30PE 150bp

75%Q30PE 150 3 days

HiSeq 4000 considerations

CLUSTERING IS VERY DIFFERENT FROM 2500– PE150 - >125 is not great*– %Q30 “passes Illumina spec”*

– ExAmp duplicates*– Need to consider how you handle duplicates

– RNA-seq is fine– Exome-seq is fine– Genomes are fine

Instruments explained - MiSeq

~600bp fragments

+/- 50bp overlap

300bp reads

Instruments explained - NextSeq

NGS QC – library prepQUALITY CONTROL OF LIBRARIES IS IMPORTANT.

TITRATION FLOWCELLS AND FAILED RUNS ARE EXPENSIVE.TRY TO IDENTIFY ISSUES BEFORE RUNNING ANY LANES.QC IS SPECIFIC TO YOUR SAMPLES.

QUANTITATION OF LIBRARIES IS IMPORTANT.SOME QC CAN ONLY BE DONE ONCE YOU HAVE GENERATED DATA

Good

Bad

Bioanalyser qPCR Analysis

NGS QC – FastQC

NGS QC – MGA

NGS QC – MGALIBRARY QC – CONTAMINANT DETECTION

SAMPLE 100,000 READS FROM FASTQREADS TRIMMED TO 36BPALIGN TO MULTIPLE GENOMES USING BOWTIE

LIBRARY QC – ADAPTER DETECTIONSAMPLE 100,000 READS FROM FASTQREADS CONVERTED TO FASTAALIGN TO “ADAPT-OME” USING EXONERATE

LIBRARY QC- YIELDCOUNT NUMBER OF READS (SINGLE-END ONLY)DISPLAY NUMBER ON A PRE-DEFINED SCALEDISPLAY LANES IN FLOWCELL CONFIGURATION

NGS QC – MGA

NGS QC – MGA

The Genomics Core sequencing services

James Hadfield NEB March 2016

The Genomics Core sequencing services

The Genomics Core sequencing services

Service metrics Jan 2016

– TAT has been 2-3 weeks (often as little as 1 week)– Most sequencing works very well, but…

The Genomics Core sequencing services

The Genomics Core sequencing services

NGS methods

A genomic case report

A genomic case report

NFKBIA S32G

SIFT: deleterious(0)PolyPhen: probably_damaging(0.979)