module 4 mcpherson 2012 - files.bioinformatics.ca · 120529 2 module’4’...

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Canadian Bioinforma2cs Workshops

www.bioinforma2cs.ca

2 Module #: Title of Module

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Module 4 Mapping and Genome Rearrangement

John McPherson, Ph.D.

ATCAA CTAAG

DNA fragment

Paired-end Reads

Module 1 bioinformatics.ca

Platform complexity

Increasing Run Time

Increasing Data

Per Run

Moving away from a “one-size-fits-all” platform

$

$

Cross-platform data integration needed.

700Mb/23h

150Mb/3h

100Mb/1h

2Gb/27h

100Gb/15d

90Gb/10d

600Gb/10d

14TB/run

120Gb/1d

Proton? GridION?

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Module bioinformatics.ca

Single Molecule

Amplified Template

Long Read

Short Read


Basecalling

•  How do we translate the machine data to base calls? •  How do we es2mate and represent sequencing errors?

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Spectral overlap

http://www.olympusfluoview.com/theory/bleedthrough.html


Spectral overlap


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Spectral overlap



Spectral overlap


Sources of error

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Sources of error Illumina: Pre-‐phasing & Phasing


What is a base quality?

Base Quality Perror(obs. base)

3 50 % 5 32 %

10 10 % 20 1 % 30 0.1 % 40 0.01 %

PHRED values: -  Sequence a known template and align reads -  determine error rate.

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Calibra@ng Base Quali2es

“Original” Recalibrated

Mark DePristo Broad Institute June 2009


Error Profiles

Roche 454 •  error rate is low (< 0.5%) •  most errors are INDELs

72%

24%

4%

insertions

deletions

substitutions

Illumina •  error rate is also low •  most errors substitions

Slides by M. Stromberg

Correct 99.5%

Error 0.5%

Error rate over all bases

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Mismatch by cycle


Fasta files ASF-1.fa ASF-2.fa

•  Reads are oWen stored in fasta files •  Separate file for forward and reverse pairs •  header line -‐-‐ read name/pairing info •  sequence line -‐-‐ nucleo2des

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Fastq files

ASF-1.fastq ASF-2.fastq

•  header line: @SEQUENCE_ID •  sequence line •  line beginning with + •  encoded quality value line

•  Most reads are stored in fastq •  4 lines per read


Alignment

•  Reference-‐based alignment: •  Goal: find posi2on in genome from which read was sampled

–  Comparison is to the human reference genome (eg HG19)

•  Can't we use BLAT or BLAST? –  op2mized for long reads –  slow

•  Things to consider: –  support for your technology –  speed / sensi2vity –  parallelism –  tolerance for gapped alignment –  handling of mul2ple good mappings

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Illumina AB SOLiD Roche 454 Helicos gapped all alignments multithreaded

Bowtie X X X X BWA X X X X BFAST X X X X X X X Corona Lite X X ELAND X GenomeMapper X X X X gnumap X X X X karma X X X * MAQ X X MOSAIK X X X X X X X MrFAST X X X MrsFAST X X Novoalign X X X * RMAP X X SeqMap X X X SHRiMP X X X X X X Slider X X SOAP2 X X X SSAHA2 X X X X SOCS X X SXOligoSearch X X X X Zoom X X * X Slides by M. Stromberg


0 5000 10000 15000 20000

Karma

Bowtie

SOAP2

BWA

ELAND2

MOSAIK

srprism

BFAST

Novoalign

speed (reads/s)

alig

ners

Performance

Illumina 37 bp (human genome) program aligned reads/s Karma 15,635 Bowtie 13,889 SOAP 13,580 BWA 10,314 ELAND2 8,859 MOSAIK 6,792 Srprism 2,768 BFAST 1,125 Novoalign 1,095


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Genome Mapping


De novo assembly

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Reference alignment


Reference alignments

?

Reference genome

Sequence read

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Reference alignments Reference genome

Sequence read

x x x


Reference alignments Reference genome

Sequence read

x x x x

?

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Alignment Quality

0

10

20

30

40

50

60

70

0 20 40 60

Act

ual a

lignm

ent q

ualit

y Assigned alignment quality

optimal MOSAIK using PE AQs Slides by M. Stromberg


Alignment Quality

alignment errors due to heuristic algorithm

probability that the best hit is wrong


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INDEL Cleaning


“You like tomato and I like tomahto”

George Gershwin •  NGS010 BRCA1 dele2on variant

…CGCTTTAATTTATTTGTG…!!…CGCTTTATTTGTG…!!

…CGC-----TTTATTTGTG…!!

…CGCTTTA-----TTTGTG…!!

!c.1500_1504delTTTAA! c.1504_1508delATTTA!

Reference

Variant

CAP/CLIA Sanger sequence NGS pipeline

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De novo assembly


De novo assembly

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De novo assembly


De novo assembly

Read from a repeat

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De novo assembly

Long Reads


What are Paired Reads?

ATCAA CTAAG

Insert size (IS)

DNA fragment

Paired-end Reads

Slides by M. Brudno

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De novo assembly


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SAM/BAM

•  SAM = text, BAM = binary

SRR013667.1 99 19 8882171 60 76M = 8882214 119 NCCAGCAGCCATAACTGGAATGGGAAATAAACACTATGTTCAAAGCAGAGAAAATAGGAGTGTGCAATAGACTTAT #>A@BABAAAAADDEGCEFDHDEDBCFDBCDBCBDCEACB>AC@CDB@>>CB?>BA:D?9>8AB685C26091:77

Read name Flag Reference Position CIGAR Mate Position

Bases

Base Qualities


CIGAR

MD:3^A03C5

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samtools

•  used for low level processing of BAM/SAM files •  convert between BAM and SAM •  sort alignments by posi2on •  create index for sorted BAM file


What kinds of varia@on is there?

•  Single Nucleo2de Polymorphisms (SNPs) •  Short indels (< read length) •  Structural varia2ons

–  large scale inser2ons and dele2ons –  Inversions –  Transloca2ons –  Copy number varia2on

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Structural variants Mate-pair and paired-end reads can be used to detect structural

variants

Fragmentation & circularization to an internal adaptor

Shear

Isolate internal adaptors and fragment ends

Mate-Pairs Paired-Ends

Fragmentation

Add amplification and sequencing adaptors

Sequence

Add amplification and sequencing adaptors

Genomic DNA

1 - 20kb 200 – 500bp


Clusters of aberrantly aligned read pairs

Mapping of read pairs to reference •  Spanning unexpected distance •  Unexpected orientation

• Detection of: • Deletions • Insertions • Translocations • Inversions

Fragment size

Fragment number

< <

Insertion

> <

Deletion

> <>Build35

<

inv Map

Seq

del Map

Seq

> <

Concordant Inversion translocation

ChrA ChrB

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Inser@on: signature

Mapped distance

Insert size Mapped distance < IS - 2.s.d

Size of insertion = Insert size - Mapped distance

don

ref

Slides by M. Brudno


Inser@on: consistency 1.  Overlap 2.  Size of inser2on explained by X = Size of inser2on explained by Y

X

Y

X

Y

don

ref

Slides by M. Brudno

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Inser@on: narrowing down the loca@on

don

ref

Possible location of insertion

•  Insertion lies within spanning region of matepair •  For clusters, it lies within the intersection

Slides by M. Brudno


SV summary

Type Mapped Distance Orienta@on

Inser2on too big correct

Dele2on too small correct

Inversion *

Tandem duplica2on *

Interchromosomal different chromosomes

N/A

Slides by M. Brudno

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Where can we go wrong: missed inser@on

don

ref

IS Insertions larger than IS cannot be detected with basic method


Soma@c vs. Germline

•  tumor vs. normal sequencing •  approach 1:

–  find SVs separately in two samples –  filter out soma2c SVs that overlap germline SVs

•  approach 2 –  find soma2c SVs –  for each soma2c SV, find any type of evidence in germline

•  a single discordant but non-‐consistent matepair?

–  filter out anything with evidence

Slides by M. Brudno

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Variant detection - distinguishing novel variants

from errors Reference: ACGT …

Germline variants 50% Ref : 50% Var 40% for : 60% rev




Strand bias

Misaligned reads - Not consistent so sometimes seen as somatic

PCR artifact Germline variants 50% Ref : 50% Var 40% for : 60% rev

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80% Ref : 20% Var

Strand bias

Misaligned reads PCR artifact Somaic mutations


Structural Variants and Split Reads

Paired Short Reads

Align

Most of these pairs can be aligned to the reference genome

For some paired-end reads one of the pair may not be mapped because it goes across the breakpoint of a structural variant. We call such reads split reads.

Slides by M. Brudno

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Split read signatures

don

ref

Deletion

don

ref

Insertion

don

ref

don

ref Slides by M. Brudno


Pair informed split mapping

ref

Deletion

reference region 1

reference region 2

•  searching the whole genome for split mappings –  gives a lot of false mappings –  too slow

•  can exactly es2mate breakpoint and indel sizes •  can detect very small dele2ons

Slides by M. Brudno

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SV SoRware and Exercise •  There are many: •  GASV (will use this today)

–  hsp://compbio.cs.brown.edu/soWware.html –  S. Sindi, E. Helman, A. Bashir, B.J. Raphael. (2009) A Geometric Approach for Classifica2on and Comparison of Structural Variants.Bioinforma*cs. 25: i222-‐i230

•  Breakdancer –  hsp://breakdancer.sourceforge.net/ –  hsp://www.nature.com/nmeth/journal/v6/n9/abs/nmeth.1363.html


Gene fusions

•  if a linking signature connects two genes, this might indicate a gene fusion

ChrA

ChrB

Gene X

Gene Y

Gene XY Protein

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Things we have set up:

•  Loaded data files to an S3 bucket •  We brought up an Ubuntu (Linux) instance, and loaded a whole bunch of soWware for NGS analysis.

•  We then cloned this, and made separate instances for everybody in the class.

•  We’ve simplified the security: you basically all have the same login and and file access, and opened ports. In your own world you would be more secure.


All on Wiki! �http://bioinformatics.ca/workshop_wiki/�"Login: FirstnameLastname�"Password: guest �

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On Mac: Control+


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http://bioinformatics.ca/workshop_wiki/��"Login: FirstnameLastname�"Password: guest �


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# is your assigned student number�


Ask your ques@on, and then gather the data, the tools and hardware you need

•  Data and Databases: you will take workshops, you will read papers, and you will go on-‐line: SeqAnswers & maybe the bioinforma2cs.ca Links Directory

•  Tools: you will take workshops, you will read papers, and you will go on-‐line: SeqAnswers & maybe the bioinforma2cs.ca Links Directory

•  Hardware: you need to decide?

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We are now going to start an exercise in mapping and structural variant

detec2on.


We are on a Coffee Break & Networking Session

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Cryp2c Fusion Oncogene

•  Welch et al. JAMA 305; 1577-‐1584 –  April 20, 2011

•  Acute Promyelocy2c Leukemia (APL) –  >90% associated with gene fusion PML-‐RARA –  Rapid diagnosis is essen2al as adding all-‐trans re2noic acid to chemotherapy leads to substan2ally improved outcome (5yr event-‐free-‐survival of 69% compared with 29% with chemotherapy alone)



•  39 year old pa2ent diagnosed with acute myeloid leukemia (AML) in first remission referred for allogenic stem cell transplanta2on.

•  Cytogene2cs indicated a poor prognosis and absence of a PML-‐RARA fusion.

•  Leukemic cytomorphology consistent with APL •  Course of treatment uncertain – APL or AML with poor prognosis?

Welch et al. JAMA 305; 1577-1584

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•  Whole genome sequencing revealed a 77kb inser2on from chromosme 15 into the second intron of the RARA gene on chromosome 17 resul2ng in a classic PML-‐RARA fusion.

•  7 week turnaround; ~$40,000 •  Pa2ent received ATRA and is in remission at 15 months.





module 4 mcpherson 2012 - files.bioinformatics.ca · 120529 2 module’4’...

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