High Throughput Sequencing Tutorial 6 High Throughput Sequencing

HTS tools and analysis Visualization - IGV Analysis platform – Galaxy Tuning up the pipelines

Working with IGV

http://www.broadinstitute.org/igv/

Why and how to work with IGV

Base qualities, comparison between samples

False positive indels

Same mapping statistics – different meaning What might cause this low percentage of mapping?

The sample contains a high percentage of contamination The sample is very different from the reference genome

One image is worth a thousand words…

Structural Variations Large deletion in the sample compared to the reference genome

Galaxy

https://main.g2.bx.psu.edu/

Use your account name and password to login to Galaxy:

Uploading data to Galaxy

Mapping, filtering and conversion to BAM

Mapping

Filter SAM file

Convert SAM to BAM

Variant calling

Create pileup

Find variants

Tuning up the pipelines

How can mapping parameters affect the results 1 mismatch per read 5 mismatches per read

False positives vs. true negatives One pipeline for all projects? False positives vs. true negatives 3-bases insertion

How can you tune your analysis? Try different programs. Mapping: Change mapping parameters Use non-unique mappings Don’t filter duplicates Variants: Change variant filtration Change variant merging – penetrance, different heredity, low coverage in one individual… Look for bigger variants: big insertions/ deletions, inversions, copy number variations etc. Gene expression: Change the test threshold