User Tools

Site Tools


assignments:ex13

Differences

This shows you the differences between two versions of the page.

Link to this comparison view

Both sides previous revision Previous revision
Next revision
Previous revision
assignments:ex13 [2017/11/15 12:40]
dokuroot
assignments:ex13 [2018/11/27 12:56]
dokuroot
Line 1: Line 1:
-=====RNA-seq Data Analysis=====+~~NOTOC~~ 
 +=====RNA-seq Data Analysis: part 1=====
 ---- ----
 The purpose of this exercise is to introduce tools for analyzing differential gene expression in RNA-seq data. You will analyze RNA-seq data from human reference (a mix of tissues) and brain tissue to identify genes for which expression is enriched in the brain. Due to time constraints,​ the data we will analyze is a subset (chr22) of a larger RNA-seq dataset. The purpose of this exercise is to introduce tools for analyzing differential gene expression in RNA-seq data. You will analyze RNA-seq data from human reference (a mix of tissues) and brain tissue to identify genes for which expression is enriched in the brain. Due to time constraints,​ the data we will analyze is a subset (chr22) of a larger RNA-seq dataset.
Line 8: Line 9:
 \\ \\
  
-**Note:** this pipeline is no longer updated and has been replaced with a more efficient and accurate pipeline, however, we will use the original Tuxedo pipeline because there is far more support currently available for it and it has fewer bugs to contend with. The new and improved pipeline consists of a similar suite of tools: HISAT, StringTie, and Ballgown. ​+**Note:** this pipeline is no longer updated and has been replaced with a more efficient and accurate pipeline, however, we will use the original Tuxedo pipeline because there is far more support currently available for it and it has fewer bugs to contend with. The new and improved pipeline consists of a similar suite of tools: HISAT, StringTie, and Ballgown. ​See https://​www.nature.com/​articles/​nprot.2016.095
  
 ====OUTLINE==== ====OUTLINE====
Line 23: Line 24:
 ====Quality control and filtering==== ====Quality control and filtering====
  
-**1. ** Open cyberduck and sftp into the montgomery lab server:\\ 
-''​montgomeryserver,​biology.colostate.edu''​ 
-\\ \\ 
  
-**2. ** Download brain and download ​the ''​brain_data'' ​folder onto your desktop\\ +**1. ** Open a terminal window ​and change into the ''​brain_data'' ​directory: ''​/​Users/​bz360/Documents/RNAseq_Files/​brain_data'' ​\\ 
-''​genomics/Documents/RNA-seq_data/​brain_data''​+
  
 There are 12 RNA-seq datasets corresponding to paired-end data for 3 replicates from two sample sets (brain and ref). Examine a few lines of one of the files using ''​zmore''​ or ''​zless''​. ​ What information is contained in each line? There are 12 RNA-seq datasets corresponding to paired-end data for 3 replicates from two sample sets (brain and ref). Examine a few lines of one of the files using ''​zmore''​ or ''​zless''​. ​ What information is contained in each line?
Line 46: Line 44:
 \\ \\
  
-**3.** Assess the quality of the data using ''​FastQC'':​+**2.** Assess the quality of the data using ''​FastQC'':​
 \\ \\
  
Line 56: Line 54:
 \\ \\
     ​     ​
-**4.** Trim adapter sequences and quality filter the RNA-seq data (fastq files) using ''​Trimmomatic'':​+**3.** Trim adapter sequences and quality filter the RNA-seq data (fastq files) using ''​Trimmomatic'':​
   ​   ​
 Trim adapter sequences and quality filter each dataset using Trimmomatic (you will run trimmomatic 6 times in total). Trim adapter sequences and quality filter each dataset using Trimmomatic (you will run trimmomatic 6 times in total).
  
-  $ trimmomatic PE -phred33 ​'​input_fastq_1'​ '​input_fastq_2'​ 'output_fastq_paired_1' 'output_fastq_unpaired_1' 'output_fastq_paired_2' 'output_fastq_unpaired_2' ILLUMINACLIP:/​usr/share/trimmomatic/​adapters/​TruSeq3-PE.fa:​2:​30:​10 LEADING:3 TRAILING:3 SLIDINGWINDOW:​4:​15 MINLEN:36 +  $ trimmomatic PE '​input_fastq_1'​ '​input_fastq_2'​ 'trimmed_P1' 'trimmed_P2' 'trimmed_U1' 'trimmed_U2' ILLUMINACLIP:/​Users/bz360/Documents/​TruSeq3-PE.fa:​2:​30:​10 LEADING:3 TRAILING:3 SLIDINGWINDOW:​4:​15 MINLEN:36 
 + 
 +For output file names, use:\\ 
 +trimmed_brain1_P1.fastq.gz\\ 
 +trimmed_brain1_P2.fastq.gz\\ 
 +trimmed_brain1_U1.fastq.gz\\ 
 +trimmed_brain1_U2.fastq.gz\\
  
 //See the Trimmomatic manual for a detailed description of options: http://​www.usadellab.org/​cms/​uploads/​supplementary/​Trimmomatic/​TrimmomaticManual_V0.32.pdf // //See the Trimmomatic manual for a detailed description of options: http://​www.usadellab.org/​cms/​uploads/​supplementary/​Trimmomatic/​TrimmomaticManual_V0.32.pdf //
 \\ \\ \\ \\
-//What proportion of the reads were retained?//+ 
 +**Submit an answer to the following question on Canvas:** 
 +\\ 
 +What proportion of the reads in each library was retained?
 \\ \\ \\ \\ \\ \\
  
-**5.** Assess the quality of one of the datasets after quality filtering using ''​FastQC'':​+**4.** Assess the quality of one of the datasets after quality filtering using ''​FastQC'':​
  
 In FastQC: ​ In FastQC: ​
   File>​open   File>​open
 +  ​
 +\\
 +
 +**5.** Create a ''​bowtie index''​ for the human chromosome 22 sequence: \\
 +
 + $ bowtie2-build '​sequence.fa'​ '​prefix'​
 +
 +The chr22 sequence should in the ''​brain_data''​ folder: ''​Homo_sapiens.GRCh38.dna.chromosome.22.fa''​.
 +\\
 +You will need to decompress the file if it has the ''​.gz''​ extension. ​ \\
 +\\
 +If you don't have the chromosome sequence, you can download it here:
 +\\
 +ftp://​ftp.ensembl.org/​pub/​release-94/​fasta/​homo_sapiens/​dna/​
 +
 +
 +For the bowtie prefix, use ''​chr22''​.\\
assignments/ex13.txt · Last modified: 2018/11/27 12:56 by dokuroot