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assignments:ex13 [2017/11/15 12:38]
dokuroot
assignments:ex13 [2018/11/27 12:56] (current)
dokuroot
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 +~~NOTOC~~
 =====RNA-seq Data Analysis: part 1===== =====RNA-seq Data Analysis: part 1=====
 ---- ----
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 \\ \\
  
-**Note:** this pipeline is no longer updated and has been replaced with a more efficient and accurate pipeline, however, we will use the original Tuxedo pipeline because there is far more support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 ​+**Note:** this pipeline is no longer updated and has been replaced with a more efficient and accurate pipeline, however, we will use the original Tuxedo pipeline because there is far more support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====
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 ====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?
 \\ \\
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   * ref_rep3_1.fastq.gz   * ref_rep3_1.fastq.gz
   * ref_rep3_2.fastq.gz   * ref_rep3_2.fastq.gz
 +\\
  
-**3.** Assess the quality of the data using ''​FastQC'':​+**2.** Assess the quality of the data using ''​FastQC'':​
 \\ \\
  
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 //See FastQC tutorial for additional details: https://​www.youtube.com/​watch?​v=bz93ReOv87Y //  ​ //See FastQC tutorial for additional details: https://​www.youtube.com/​watch?​v=bz93ReOv87Y //  ​
 +
 \\ \\
     ​     ​
-**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?// 
 \\ \\ \\ \\
  
-**5.** Assess the quality of one of the datasets after quality filtering using ''​FastQC'':​+**Submit an answer to the following question on Canvas:** 
 +\\ 
 +What proportion of the reads in each library was retained? 
 +\\ \\ \\ 
 + 
 +**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/ex13.1510774718.txt.gz · Last modified: 2017/11/15 12:38 by dokuroot