Extracting relevant information from UHTS data: analysis pipelines - - PowerPoint PPT Presentation

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Extracting relevant information from UHTS data: analysis pipelines (smallRNA) Patricia Otten 3th July 2012 JOBIM Rennes - France Fasteris SA: Illumina sequencing - founded in 2003 by L. FARINELLI and M. OSTERAS - 2012: about 20

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Extracting relevant information from UHTS data: analysis pipelines (smallRNA)

Patricia Otten 3th July 2012

JOBIM

Rennes - France

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3th July 2012 JOBIM - Rennes 2

founded in 2003 by L. FARINELLI and M. OSTERAS
2012: about 20 collaborators
capillary and UHTS sequencing + bioinformatics
private and academic labs
no business plan, no external investors, no sales forces

Fasteris SA: Illumina sequencing

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3th July 2012 JOBIM - Rennes 3

Key technology based on the concept of DNA colonies, invented in 1996 at the GlaxoWellcome's Geneva Biomedical Research Institute

Mayer P., Farinelli L. and Kawashima, E., 1997, Patent application WO 98/44151

Illumina sequencing

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3th July 2012 JOBIM - Rennes 4

Library preparation (smallRNA protocol)

3 ug total RNA selection of small RNAs (20-30 nt) acrylamide gel purification single-stranded ligation

f the 3' adapter

single-stranded ligation

f the 5' adapter

reverse transcription, PCR, index addition, gel purification

P7

P5

Illumina sequencing: step1

index

library

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3th July 2012 JOBIM - Rennes 5

Templates are hybridized to a surface (flowcell) and in situ amplified (bridge amplification) to form DNA colonies.

each colony produces one read
all colonies are sequenced in parallel
~150 mio passed filter reads per lane

Illumina sequencing: step2

x

Flowcell preparation

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3th July 2012 JOBIM - Rennes 6

Illumina sequencing: step 3

Incorporation of reversible- terminator nucleotides labeled with fluorescent dyes

base per base sequencing (50, 100 cycles, SR or PE)
laser excitation and image capture; release of dye;
intensities extraction and base calling by RTA software

1x100 run: 1 week; 1.5 TB intensities; 200 GB sequences;

Sequencing

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3th July 2012 JOBIM - Rennes 7

Adapter trimming

Trimming (smallRNAs)

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3th July 2012 JOBIM - Rennes 8

Introduction to smallRNAs

chemical modifications of

ther RNAs, mainly rRNAs,

tRNAs and snRNAs RNA splicing, guides for telomere elongation translation from dsRNA downregulation transposons silencing poorly conserved downregulation of genes highly conserved

Sequencing by siRNA: a novel generic tool for virus discovery

Kreuze et al. (2009) Complete viral genome sequence and discovery of

novel viruses by deep sequencing of small RNAs: a generic method for diagnosis, discovery and sequencing of viruses. Virology 388: 1-7

Expression analysis Virus assembly

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3th July 2012 JOBIM - Rennes 9

Introduction to smallRNAs

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3th July 2012 JOBIM - Rennes 10

Pipelines and automation → automation + checks → handle unexpected issues, keep time for the client → a pipeline is a set of predetermined tasks that have to be executed to complete a specific analysis produce meaningfull data time resources

www.photo-dictionary.com

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3th July 2012 JOBIM - Rennes 11

Pipelines and automation Eg: comparison of libraries in terms of miRNA coverage mapping (ref. genome) miRNA coverage normalization library comparison visualization mapping (ref. genome) Makefiles Bash scripts R scripts

1. reference
2. indexing
3. mapping
4. format conversion
5. reporting

Each module may involve one

r several processes

insert selection

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3th July 2012 JOBIM - Rennes 12

reads Expression pipelines inserts mapping (ref. genome) sequence profile annotated features peak detection mapping (sequence db) post-processing coverage

BWA Bash R Seqmonk Bedtools Bash/R Perl PMRD mirBase iGenome

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3th July 2012 JOBIM - Rennes 13

reads Expression inserts mapping (ref. genome) sequence profile annotated features peak detection mapping (sequence db) post-processing coverage

BWA Bash R Seqmonk Bedtools Bash/R Perl PMRD mirBase iGenome

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3th July 2012 JOBIM - Rennes 14

reads Expression inserts mapping (ref. genome) sequence profile annotated features peak detection mapping (sequence db) post-processing coverage

BWA Bash R Seqmonk Bedtools Bash/R Perl PMRD mirBase iGenome

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3th July 2012 JOBIM - Rennes 15

reads Expression inserts mapping (ref. genome) sequence profile annotated features peak detection mapping (sequence db) post-processing coverage

BWA Bash R Seqmonk Bedtools Bash/R Perl PMRD mirBase iGenome

23 24 25 26 27 28 29

150000
100000
50000

50000 100000 150000 200000 250000

Size profile, BDGP5.25

LIB-1

Insert size RPM

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reads Expression inserts mapping (ref. genome) sequence profile annotated features peak detection mapping (sequence db) post-processing coverage

BWA Bash R Seqmonk Bedtools Bash/R Perl PMRD mirBase iGenome

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3th July 2012 JOBIM - Rennes 17

reads Expression inserts mapping (ref. genome) sequence profile annotated features peak detection mapping (sequence db) post-processing coverage

BWA Bash R Seqmonk Bedtools Bash/R Perl PMRD mirBase iGenome

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3th July 2012 JOBIM - Rennes 18

reads Expression inserts mapping (ref. genome) sequence profile annotated features peak detection mapping (sequence db) post-processing coverage

BWA Bash R Seqmonk Bedtools Bash/R Perl PMRD mirBase iGenome

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3th July 2012 JOBIM - Rennes 19

reads Expression inserts mapping (ref. genome) sequence profile annotated features peak detection mapping (sequence db) post-processing coverage

BWA Bash R Seqmonk Bedtools Bash/R Perl PMRD mirBase iGenome count insertNb [M] * probeLength [K] RPKM =

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3th July 2012 JOBIM - Rennes 20

reads Expression inserts mapping (ref. genome) sequence profile annotated features peak detection mapping (sequence db) post-processing coverage

BWA Bash R Seqmonk Bedtools Bash/R Perl PMRD mirBase iGenome

0.8 0.9 0.95 0.99

Comparison scores between pairs of libraries. n1,n2~binomial distribution with same probability of event (p=(n1/N2+n2/N2)/2); score~p(observing a count <n1 or >n2)

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3th July 2012 JOBIM - Rennes 21

reads Expression inserts mapping (ref. genome) sequence profile annotated features peak detection mapping (sequence db) post-processing coverage

BWA Bash R Seqmonk Bedtools Bash/R Perl PMRD mirBase iGenome

s

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3th July 2012 JOBIM - Rennes 22

Virus identification

SiRNAs:

class of dsRNAs of 20-25 nts
involved in post-transcriptional gene silencing
endogenous or exogenous

→ synthetic dsRNA introduced into cells can induce silencing of specific

genes of interest → viral infection: presence of viral dsRNA leading to siRNAs that participate in the cell antiviral response;

Sequencing by siRNA: a novel generic tool for virus discovery

Kreuze et al. (2009) Complete viral genome sequence and discovery of

novel viruses by deep sequencing of small RNAs: a generic method for diagnosis, discovery and sequencing of viruses. Virology 388: 1-7

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3th July 2012 JOBIM - Rennes 23

reads (infected sample) Virus assembly pipelines inserts mapping (host ref. genome) mapping (host contigs) mapping (viral db) de novo assembly (unmapped inserts) reads (control sample) inserts de novo assembly

Perl/R Perl/R BWA Velvet+Oases Velvet+Oases Blast/BWA/Mummer Velvet+Oases iGenome RefSeq

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3th July 2012 JOBIM - Rennes 24

reads (infected library) Virus assembly inserts mapping (host ref. genome) mapping (host contigs) mapping (viral db) de novo assembly (unmapped inserts) reads (control library) inserts de novo assembly

Perl Perl BWA Velvet+Oases Velvet+Oases Blast/BWA/Mummer Velvet+Oases iGenome RefSeq

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3th July 2012 JOBIM - Rennes 25

reads (infected library) Virus assembly inserts mapping (host ref. genome) mapping (host contigs) mapping (viral db) de novo assembly (unmapped inserts) reads (control library) inserts de novo assembly

Perl Perl BWA Velvet+Oases Velvet+Oases Blast/BWA/Mummer Velvet+Oases iGenome RefSeq

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3th July 2012 JOBIM - Rennes 26

reads (infected library) Virus assembly inserts mapping (host ref. genome) mapping (host contigs) mapping (viral db) de novo assembly (unmapped inserts) reads (control library) inserts de novo assembly

Perl Perl BWA Velvet+Oases Velvet+Oases Blast/BWA/Mummer Velvet+Oases iGenome RefSeq

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3th July 2012 JOBIM - Rennes 27

reads (infected library) Virus assembly inserts mapping (host ref. genome) mapping (host contigs) mapping (viral db) de novo assembly (unmapped inserts) reads (control library) inserts de novo assembly

Perl Perl BWA Velvet+Oases Velvet+Oases Blast/BWA/Mummer Velvet+Oases iGenome RefSeq

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3th July 2012 JOBIM - Rennes 28

Extracting relevant information from UHTS data: analysis pipelines (smallRNA)

JOBIM

Rennes - France

Fasteris SA: Illumina sequencing

Key technology based on the concept of DNA colonies, invented in 1996 at the GlaxoWellcome's Geneva Biomedical Research Institute

Illumina sequencing

Library preparation (smallRNA protocol)

3 ug total RNA selection of small RNAs (20-30 nt) acrylamide gel purification single-stranded ligation

single-stranded ligation

reverse transcription, PCR, index addition, gel purification

P7

P5

Illumina sequencing: step1

index

library

Templates are hybridized to a surface (flowcell) and in situ amplified (bridge amplification) to form DNA colonies.

Illumina sequencing: step2

x

Flowcell preparation

Illumina sequencing: step 3

Incorporation of reversible- terminator nucleotides labeled with fluorescent dyes

1x100 run: 1 week; 1.5 TB intensities; 200 GB sequences;

Sequencing

Adapter trimming

Trimming (smallRNAs)

Introduction to smallRNAs

Sequencing by siRNA: a novel generic tool for virus discovery

Kreuze et al. (2009) Complete viral genome sequence and discovery of

Expression analysis Virus assembly

Introduction to smallRNAs

Pipelines and automation → automation + checks → handle unexpected issues, keep time for the client → a pipeline is a set of predetermined tasks that have to be executed to complete a specific analysis produce meaningfull data time resources

Pipelines and automation Eg: comparison of libraries in terms of miRNA coverage mapping (ref. genome) miRNA coverage normalization library comparison visualization mapping (ref. genome) Makefiles Bash scripts R scripts

Each module may involve one

insert selection

reads Expression pipelines inserts mapping (ref. genome) sequence profile annotated features peak detection mapping (sequence db) post-processing coverage

BWA Bash R Seqmonk Bedtools Bash/R Perl PMRD mirBase iGenome

reads Expression inserts mapping (ref. genome) sequence profile annotated features peak detection mapping (sequence db) post-processing coverage

BWA Bash R Seqmonk Bedtools Bash/R Perl PMRD mirBase iGenome

reads Expression inserts mapping (ref. genome) sequence profile annotated features peak detection mapping (sequence db) post-processing coverage

BWA Bash R Seqmonk Bedtools Bash/R Perl PMRD mirBase iGenome

reads Expression inserts mapping (ref. genome) sequence profile annotated features peak detection mapping (sequence db) post-processing coverage

BWA Bash R Seqmonk Bedtools Bash/R Perl PMRD mirBase iGenome

reads Expression inserts mapping (ref. genome) sequence profile annotated features peak detection mapping (sequence db) post-processing coverage

BWA Bash R Seqmonk Bedtools Bash/R Perl PMRD mirBase iGenome

reads Expression inserts mapping (ref. genome) sequence profile annotated features peak detection mapping (sequence db) post-processing coverage

BWA Bash R Seqmonk Bedtools Bash/R Perl PMRD mirBase iGenome

reads Expression inserts mapping (ref. genome) sequence profile annotated features peak detection mapping (sequence db) post-processing coverage

BWA Bash R Seqmonk Bedtools Bash/R Perl PMRD mirBase iGenome

reads Expression inserts mapping (ref. genome) sequence profile annotated features peak detection mapping (sequence db) post-processing coverage

BWA Bash R Seqmonk Bedtools Bash/R Perl PMRD mirBase iGenome count insertNb [M] * probeLength [K] RPKM =

reads Expression inserts mapping (ref. genome) sequence profile annotated features peak detection mapping (sequence db) post-processing coverage

BWA Bash R Seqmonk Bedtools Bash/R Perl PMRD mirBase iGenome

Comparison scores between pairs of libraries. n1,n2~binomial distribution with same probability of event (p=(n1/N2+n2/N2)/2); score~p(observing a count <n1 or >n2)

reads Expression inserts mapping (ref. genome) sequence profile annotated features peak detection mapping (sequence db) post-processing coverage

BWA Bash R Seqmonk Bedtools Bash/R Perl PMRD mirBase iGenome

s

Virus identification

SiRNAs:

→ synthetic dsRNA introduced into cells can induce silencing of specific

genes of interest → viral infection: presence of viral dsRNA leading to siRNAs that participate in the cell antiviral response;

Sequencing by siRNA: a novel generic tool for virus discovery

Kreuze et al. (2009) Complete viral genome sequence and discovery of

reads (infected sample) Virus assembly pipelines inserts mapping (host ref. genome) mapping (host contigs) mapping (viral db) de novo assembly (unmapped inserts) reads (control sample) inserts de novo assembly

Perl/R Perl/R BWA Velvet+Oases Velvet+Oases Blast/BWA/Mummer Velvet+Oases iGenome RefSeq

reads (infected library) Virus assembly inserts mapping (host ref. genome) mapping (host contigs) mapping (viral db) de novo assembly (unmapped inserts) reads (control library) inserts de novo assembly

Perl Perl BWA Velvet+Oases Velvet+Oases Blast/BWA/Mummer Velvet+Oases iGenome RefSeq

reads (infected library) Virus assembly inserts mapping (host ref. genome) mapping (host contigs) mapping (viral db) de novo assembly (unmapped inserts) reads (control library) inserts de novo assembly

Perl Perl BWA Velvet+Oases Velvet+Oases Blast/BWA/Mummer Velvet+Oases iGenome RefSeq

reads (infected library) Virus assembly inserts mapping (host ref. genome) mapping (host contigs) mapping (viral db) de novo assembly (unmapped inserts) reads (control library) inserts de novo assembly

Perl Perl BWA Velvet+Oases Velvet+Oases Blast/BWA/Mummer Velvet+Oases iGenome RefSeq

reads (infected library) Virus assembly inserts mapping (host ref. genome) mapping (host contigs) mapping (viral db) de novo assembly (unmapped inserts) reads (control library) inserts de novo assembly

Perl Perl BWA Velvet+Oases Velvet+Oases Blast/BWA/Mummer Velvet+Oases iGenome RefSeq

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