Trimming and Assessing Data

Bioinformatics Pipeline: Get DNA of interest–> Sequence–>Raw Data–>Trim Data, Assess Overall Quality–> Assemble Data–>Quality Assessment–>Annotation–>Data Interpretation

What us Trimming

  • Trimming is a pre-processing step that removes poor quality data or technical sequences, like adapters
  • Poor data can cause sub-optimal analyses later down the pipeline
  • Trimmomatic is air-aware and is optimized for Illumina NGS data

Removing Technical (adapter) Sequences Using Trimmomatic

  • Trimmomatic uses two methods to remove technical sequences

Simple Mode: Scans each read from 5′ end to 3′ end and looks for under provided adapters. The user provided adapter is something you specify in the command line when setting up the run. A local alignment is used and if the alignment score exceed the user defined threshold, the aligned region

-Palindrome Mode: This mode is optimized for “adapter read-through”

Screen Shot 2018-02-27 at 8.30.28 AM.png

 

Screen Shot 2018-02-27 at 8.32.54 AM.png

Adapters:

  • With the data we looked at it appears that the adapter are removed at an earlier step possibly during demuiliplexing
  • FastQC does find adapter sequences on our reads before running trimmomatic
  • Still need to give Trimmomatic relevant adapter sequences so it can remove any that might be present
  • use the NexteraPE-PE.fa option, which was given from UNH
  • Could be something that goes wrong during project

What is FastQC?

  • A program that will read through the raw sequence files (FASTQ) and perform a quality check on the data. It will produce a report that summarizes the  information.
  • The best time to run FastQC is after Trimmomatic, which will clean up a lot of issues that are otherwise flagged by FastQC

 

 

 

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