/ngs_data_analysis

NGS Data Analysis Practical Project

Primary LanguageShell

Project details

1- Data Download

Download ~5M Human RNA-Seq fragements from the SRA. Link to the suggested data: https://www.ncbi.nlm.nih.gov/sra/?term=SRR8797509

2- Prepare the data

  • Construct 5 Samples, each sample contains two parts.
    • Part 1: 1M reads from the main reads file. (split the main file into 5 parts)
    • Part 2: Shuffle the main reads file, and take random 1M Reads. (split the shuffled file into 5 parts)
    • Example: Sample 1 will be divided into S1_1 & S1_2, unshuffled and shuffled, respectively.

3- FASTQ Quality Control

  • For Sample 1 only, use FASTQC to report the difference between S1_1 and S1_2

4- Trimming

  • For All Samples , Apply:
    • Mild Trimming for SX_1. {shuffled}
    • Aggressive Trimming for SX_2. {unshuffled}

5- Alignment

  • Align all the samples (1:5) using BWA and Hisat against the human reference file.
    • BWA for SX_1 and HISAT for SX_2
  • Export some useful statistics report for each sample indvidually.

6- Assembly

  • Apply reference-based trasncriptome assembly using stringTie.
    • Step1. For the 5 samples unshuffled.
    • Step2. For the 5 samples shuffled.

7- Using GTF-Compare to Compare the Generated Annotation Files to a Reference Annotation.

8- Apply Differential Expression.