Alexis Dereeper, François Sabot

Alexis Dereeper, François Sabot

Analysis of NGS raw data with Galaxy

Cleaning, data control, alignment, polymorphism

Alexis Dereeper CIBA courses – Brasil 2011

Aim of the Tutorial classes:

1- Galaxy vs Command line

2- Understand FASTQ files

3- Cleaning of Illumina data (FASTQ)

4- Perform an assembly

5- Perform a mapping of Illumina reads on a reference sequence

6- Cleaning of a multiple SAM file

Alexis Dereeper, François SabotAlexis Dereeper CIBA courses – Brasil 2011

1- Galaxy

Serveur principal: http://main.g2.bx.psu.edu/

CIRAD Server : http://gohelle.cirad.fr/galaxy/


TOOLS DATA


WEB APPLICATION - “Click'n'Play” system- transparent for user



MODULAR- Numerous default bricks (already integrated)- Adding of customizable bricks




MULTIPLE- Based on a web server (Apache...)- On a single machine, or a cluster...





BUT- Simple support- Much less powerful than terminal- Only for routine analysis - Only for limited data


MULTIPLE- Based on a web server (Apache...)- On a single machine, or a cluster...



CONNECTION FOR THE TUTORIAL CLASSES:

http://gohelle.cirad.fr/galaxy/


Connecting...


Add data...


Import data from Galaxy libraries


FASTQ file → TEXT file

STRUCTURE:

@HWUSI-EAS454_0006:1:112:14105:5498#CTTGTACGCCAAGAAGTGTAGCAAAACGGCAGAGCTCGTGGATTAAACAAACAGAGGATTTCGGTGAGGATTGAGGGGGAGT

+cfffcfeffdeefefffcffffffffcffeffffdffffafcfffffdffffdfefeddf^eececfffdfcbffb

@HWUSI-EAS454_0006:1:37:16314:3410#CTTGTAAGTGTAGCAAAACGGCAGAGCTCGTGGATTAAACAAACAGAGGATTTCGGTGAGGATTGAGGGGGAGTGGTGGCCG

+`bTbbccccceeeeeceeeecccYeedded`ceec]dddde^a`deeeec\`dddcbaadadYd`]]Jc_^bc^^\


@HWUSI-EAS454_0006:1:112:14105:5498#CTTGTA

CGCCAAGAAGTGTAGCAAAACGGCAGAGCTCGTGGATTAAACAAACAGAGGATTTCGGTGAGGATTGAGGGGGAGT

+

cfffcfeffdeefefffcffffffffcffeffffdffffafcfffffdffffdfefeddf^eececfffdfcbffb


@HWUSI-EAS454_0006:1:112:14105:5498#CTTGTA


+


SEQUENCE NAME


IUPAC SEQUENCE


@HWUSI-EAS454_0006:1:112:14105:5498#CTTGTA


+



@HWUSI-EAS454_0006:1:112:14105:5498#CTTGTA


+


Quality in ASCII


@HWUSI-EAS454_0006:1:112:14105:5498#CTTGTA


+



@HWUSI-EAS454_0006:1:112:14105:5498#CTTGTA




f → Quality = 38 (102 – 64)


@HWUSI-EAS454_0006:1:112:14105:5498#CTTGTA




WHAT IS QUALITY ?

Quality value Q is an integer mapping of p (i.e., the probability that the corresponding base call is incorrect).


FASTQC: quality control

http://www.bioinformatics.bbsrc.ac.uk/projects/download.html#fastqc


http://www.bioinformatics.bbsrc.ac.uk/projects/download.html#fastqc

Why do we need to clean ?

To remove remaining adapters/primers andlow quality sequences

→ CutAdapt



20

7


70

Your data are now ready to be analyzed...


Concatenate files


Untested Tools → NGS → Assembly → Assemble with MIRA


BLAST of putative contigs against reference


Separate sequences by original individuals RC1, RC2...



Use of regular expression via Galaxy:

→ RC[13456789] & remove reads => keep RC2

→ RC[123456789]_ & remove reads => keep RC10



Mapping: Map 'pair-end‘ reads on a reference

1- Compute positions for each read





2- Associate positions of each member of the pair






3- Selection of the more probable position respecting the conditions






3- Select of the more probable position respecting the conditions

4- Edit a SAM output file


Reference From History: Shared data/Formation/PreProcess/reference.fasta

Library: Paired-end

FASTQ files: From your history

BWA setting to use: Commonly Used

Unselect “Suppress the header in the output SAM file”

Click


SAM output file (Sequence Alignment/Map)



Sort of SAM file by coordinate


Creation of Workflow for automated analysis


Workflow: how to avoid to run all the process by hand


Alexis Dereeper, François Sabot

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