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

Whole Genome Sequencing

Tips before you start

• if possible, use haploid genome (see bee genome project where they used haploid drones)
• next best thing: highly inbred, nearly homozygous lines

Sometimes you have unexpected treasures: There is probably (almost)-homozygous cow: http://en.wikipedia.org/wiki/Chillingham_cattle

• there are cases where extra-chromosomal DNA (40-70 chloroplasts per plant cell are often in 150kb range, with 40-70 copies per organelle) contributes non-trivial portion of total DNA. Select tissues/stages with less multiple copy DNAs