Adobe Systems Adobe Systems
Moderní metody analýzy genomu
- analýza
Mgr. Nikola Tom
Brno, 22.4.2015

Deep-seq Workflow/Pipeline
workflow_deep_seq.png


GATK_best_practices.png
GATK Workflow/Pipeline
https://www.broadinstitute.org/gatk/guide/best-practices

Raw sequence = fastq
•Biological sequence
•Corresponding quality scores
•ASCII character
•(fasta+ qual, csfasta + csqual, sff)
@
SEQ_ID GATTTGGGGTTCAAAGCAGTATCGATCAAATAGTAAATCCATTTGTTCAACTCACAGTTT
+
 !''*((((***+))%%%++)(%%%%).1***-+*''))**55CCF>>>>>>CCCCCCC65

FastQC



Cutadapt
•Adaptor trimming (miRNA)
•Quality filtering
•Length filtering

Read mapping => SAM, BAM
•Usually mapping reads on reference
•miRNA - special case
–Grouping and annotate against mirBase
•DNA
–BWA, Bowtie, Bfast, SHRiMP, CLC
•RNA
–TopHat (de novo splice aligner)
•Commercial
–CLC Genomics Workbench
•De novo assembly – unknown genomes
–

Alignment



SAM



Mapping, Coverage reports
•Important checkout for lab protocol
•Specificity of PCR
•Normalization
•Settings of variant calling threshold, CNV

SNV and small InDel Calling
•Coverage
•Frequency
•Base quality
•!!!
•Genomic context (homopolymers)
•Nucleotide type
•Position in read (errors at the read end)
•Alignment errors

Structural variations
•Mate-pair library
•Long InDel
•Translocation

Annotating and filtering
•Gene
•Transcript
•dbSNP
•Regulation
•Comparative genomics
•Repeats
•Functional
•Gene ontology
•miRNA targets
•Etc.