BIOTECH-7005-BIOINF-3000

Assignment 4 [30 marks]

Due before 5pm, Friday 13th September

Assignment overview [5 marks for general format]

This assignment is based on the Alignmen/NGS practicals.

Your answers to all questions should be submitted to myUni as a .zip file containing three bash scripts (see below for details), and a single file containing the answers to the questions 3,4 and 5 (can be in any readable format, but plain text or PDF are preferred). [1 mark]

The .zip filename must start with your student number [1 mark] and your bash script must be able to run without errors. This means it should run regardless of where it is located in the directory tree. Meaningful comments are strongly advised [2 marks]

For all scripts, please use the directory ~/Assignment_4 as the parent directory for all downloads and analysis. You will be expected to hard code this into your script. All other folders must be created as subdirectories of this. It will be simplest for you to stick with the structure in NGS_skeleton [1 mark]

Practical questions [20 marks]

Scripts and associated questions

1. Script 1 must do the following:
   • Download the genomic sequence (i.e. fasta file) and annotation (i.e. gff file) of the model plant Arabidopsis thaliana to the subdirectory Refs/Athaliana from the Ensembl ftp directory (link below) [2 marks]
   • Identify how many chromosomes are in the genome, and write this information to standard output (stdout) [2 marks]
   • Identify how many unique genes are located in the genome, and send this information to stdout? [2 marks]
   • Create a genome index for use with bwa[1 marks]
2. Script 2 must do the following: download the sequencing data contained at the following links (using curl or wget) to the directory 01_rawData/fastq (note that the filename in the Box link is not informative; make sure you rename your files as below()): [2 marks]
   • SRR5882797_10M_1.fastq.gz https://universityofadelaide.box.com/shared/static/egl3n16r0ziaxlvbs9074xqd1liktnuz.gz
   • SRR5882797_10M_2.fastq.gz https://universityofadelaide.box.com/shared/static/g2ly4kzz1blus5juy426i37zl45o38pu.gz
   • Trim your data for poor quality bases and remove adapters using cutadapt. Write your output to the directory 02_trimmedData/fastq [3 marks]
   • Align paired-end reads to the genome index using bwa mem, resulting in a single .bam file in the directory 03_alignedData/bam. Ensure there are no intermediary .sam files saved. [3 marks]
   • Sort and index your bam file [2 marks]
3. Script 3 must export the following information as a tab-delimited file to the parent directory. Include the category as the first column, and the numbers as the second column
   • How many reads were mapped in total? [1 marks]
   • How many reads were mapped as a pair? [1 marks]
   • How many reads were mapped as a “proper” pair? [1 marks]

Theory questions [5 marks]

1. What is the difference between an index and a barcode? [2 marks]
2. What is the difference between a SAM file and a BAM file? [1 marks]
3. What does the CIGAR string “26M2I78M” mean? [2 marks]

Resources

• The genome (fasta) for the model plant Arabidopsis thaliana can be found here: http://ftp.ebi.ac.uk/ensemblgenomes/pub/release-51/plants/fasta/arabidopsis_thaliana/dna/ and the annotation (gff3) files can be found here: http://ftp.ebi.ac.uk/ensemblgenomes/pub/release-51/plants/gff3/arabidopsis_thaliana/. There are multiple files in these directories, so take care to choose the full genome sequence and full genome gff files.
• The genome file should be “Arabidopsis_thaliana.TAIR10.dna.toplevel.fa.gz” and not contain an “rm” or “sm” in the filename. These refer to the genome being “repeat-masked” i.e. all the repetitive elements have been ignored, or “soft-masked” were the repeats and low-complexity regions are in lower-case letters (acgt not ACGT)