User talk:Darek Kedra/sandbox 29

EMBO Tunis 2014

From sequencing data to knowledge

00 Programs used

sequence pre-processing

• SRA_toolkit ver current
• FastQC ver 0.11.2
• Trimmomatic ver 0.32
• TagDust ver 2.13
• Coral ver 1.4

general tools

• fastx_toolkit ver 0.0.13
• Samtools classic ver 0.1.19
• samtools/HTSlib ver 1.0
• Picard ver 1.119

mappers

• BWA ver 0.7.10
• LAST ver 475
• Stampy stampy-1.0.23r2059.tgz (optional)

Splice reader mappings

• fqgrep Github version plus
• TRE_library ver 0.80

viewers

• IGV ver 2.3.34
• igvtools ver 2.3.32

quantification

• NGSUtils ver 0.5.6
• HTSeq ver 0.6.1 (requires NumPy)

SNPs discovery

• GATK ver 3.2-2

01 Data files used

FASTQ files

L.amazonensis RNA-Seq

• http://www.ebi.ac.uk/ena/data/view/SRP016502

L mexicana genomic DNA

• http://www.ebi.ac.uk/ena/data/view/ERX280624

(extra set) L.enriettii genomic DNA

• http://www.ebi.ac.uk/ena/data/view/SRR835620

Stuff to read / compare

File formats

• http://biobits.org/samtools_primer.html (file formats)

• http://www.slideshare.net/lindenb/ngsformats

• http://wiki.bits.vib.be/index.php/Next-generation_sequencing

VCF

• http://www.1000genomes.org/node/101 (VCF)
• http://en.wikipedia.org/wiki/Variant_Call_Format

• http://vcftools.sourceforge.net/ (VCFTools)

BED

• http://genome.ucsc.edu/FAQ/FAQformat.html#format1
• http://www.broadinstitute.org/igv/BED
• http://www.ensembl.org/info/website/upload/bed.html
• http://bedtools.readthedocs.org/en/latest/ BEDTOOLS

GFF / GTF

• http://www.ensembl.org/info/website/upload/gff.html
• http://www.sequenceontology.org/gff3.shtml

Genomes and annotations

• L mexicana

http://tritrypdb.org/common/downloads/release-8.0/LmexicanaMHOMGT2001U1103/fasta/data/TriTrypDB-8.0_LmexicanaMHOMGT2001U1103_Genome.fasta

http://tritrypdb.org/common/downloads/release-8.0/LmexicanaMHOMGT2001U1103/gff/data/TriTrypDB-8.0_LmexicanaMHOMGT2001U1103.gff

• L.amazonensis

http://tritrypdb.org/common/downloads/release-8.0/LamazonensisMHOMBR71973M2269/fasta/data/TriTrypDB-8.0_LamazonensisMHOMBR71973M2269_Genome.fasta

• L.enriettii

http://tritrypdb.org/common/downloads/release-8.0/LenriettiiLEM3045/fasta/data/TriTrypDB-8.0_LenriettiiLEM3045_Genome.fasta

• L.major

http://tritrypdb.org/common/downloads/release-8.0/LmajorFriedlin/fasta/data/TriTrypDB-8.0_LmajorFriedlin_Genome.fasta

Extra material

(optional) Stampy

Stampy is a quite slow but at times more accurate mapper, allowing for improvement over simple BWA mappings. The basic usage is as follows:

#creating two special index files 

stampy.py --species=Lmex --assembly=Lmex_toyasembly -G Lmex_toygenome Lmex_genome.nfix.fa
#Result: 
Lmex_toygenome.stidx

stampy.py -g Lmex_toygenome -H Lmex_toyasembly   
#Result:
Lmex_toyasembly.sthash

#remapping reads already mapped with BWA (prefered option)
stampy.py -g Lmex_toygenome -h Lmex_toyasembly -t2 --bamkeepgoodreads -M LmxM.01_ERR307343_12.Lmex.bwa_mem.Lmex.bam  > LmxM.01_ERR307343_12.Lmex.bwa_mem.Lmex.stampy.sam

#convert SAM to BAM, sort and index BAM file:
java -jar ~/soft/picard_1.119/SortSam.jar \
I=LmxM.01_ERR307343_12.Lmex.bwa_mem.Lmex.stampy.sam \ O=LmxM.01_ERR307343_12.Lmex.bwa_mem.Lmex.stampy.bam \
SO=coordinate VALIDATION_STRINGENCY=SILENT CREATE_INDEX=true

#Result:
LmxM.01_ERR307343_12.Lmex.bwa_mem.Lmex.stampy.bam
LmxM.01_ERR307343_12.Lmex.bwa_mem.Lmex.stampy.bam

Mapping looking worse with this data than bwa

Quantifications of mapped reads

• Gene quantifications (DNA & RNA levels)