Biotech7005

Assignment 4 [34 marks]

Due before 5pm, Monday 23rd September

Your answers to all questions should be submitted to myUni as a .zip file containing three bash scripts, and a single file containing the answers to the questions 3,4 and 5 (can be in any readable format). [1 marks]

The .zip filename must start with your student number [1 marks] and your bash script must be able to run without errors. Meaningful comments are strongly advised [1 mark]

For all scripts, please use the directory ~/Assignment4 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. [1 mark]

Practical questions [25 marks]

1. Write a script to:
   • 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 [1 marks]
2. Write a second script to download the sequencing data contained at these links (using curl or wget) to the directory 01_rawData/fastq: [2 marks]
   • SRR5882792_10M_1.fastq.gz
   • SRR5882792_10M_2.fastq.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. Write a third script to 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]
   • Find the Arabidopsis thaliana gene associated with your ID below and identify how many inserts mapped to that region [5 marks]

Theoretical 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]

Genes For Question 3

If your student number is not listed, please contact Dan to ensure you are added to the list

ID	Gene
a1762813	AT5G39980
a1202401	AT3G19540
a1776494	AT3G27570
a1778618	AT4G05470
a1705074	AT5G42410
a1790457	AT1G63340
a1758382	AT4G14350
a1789913	AT5G55530
a1776998	AT1G78670
a1776430	AT5G66320
a1770716	AT4G13130
a1739682	AT1G62450
a1758693	AT1G20720
a1781987	AT5G45000
a1768114	AT2G36210
a1700271	AT4G18590
a1646510	AT1G72390
a1779487	AT4G35987
a1778287	AT1G49700
a1783535	AT3G12990
a1789486	AT1G47640
a1758223	AT3G23175
a1769266	AT5G61580
a1771465	AT3G28674
a1768183	AT1G48770
a1781873	AT1G03390
a1755783	AT3G12210
a1679475	AT1G49100
a1662216	AT2G23540
a1780859	AT3G29180
a1775769	AT1G55270

Resources

• The genome (fasta) and annotation (gff) files for the model plant Arabidopsis thaliana can be found here: ftp://ftp.ensemblgenomes.org/pub/plants/current/fasta/arabidopsis_thaliana and ftp://ftp.ensemblgenomes.org/pub/plants/current/gff3/arabidopsis_thaliana. The are multiple versions of the same file, so take care to choose the right version to use.
• 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)