Functional Genomics Research Stream. Computational Meeting: March 29, 2012 RNA-seq Analysis Pipeline

Transcription

1 Functional Genomics Research Stream Computational Meeting: March 29, 2012 RNA-seq Analysis Pipeline

2 CHAPTER 2 Prepare Whole Transcriptome Libraries Fragment the whole transcriptome RNA µg poly(a) RNA or ng rrna-depleted total RNA Fragment the RNA (page 14) Clean up the RNA (page 15) Fragmented RNA Assess the yield and size distribution of the fragmented RNA (page 16) Construct the amplified whole transcriptome library Hybridize and ligate the RNA (page 18) + NNNNN B NNNNN NNNNN B NNNNN Perform reverse transcription (page 19) Purify the cdna (page 20) Size select the cdna (page 21) cdna 5 PCR Primer Amplify the cdna (page 25)

3 Fragment the RNA (page 14) Clean up the RNA (page 15) Fragmented RNA Assess the yield and size distribution of the fragmented RNA (page 16) Construct the amplified whole transcriptome library Hybridize and ligate the RNA (page 18) + NNNNN B NNNNN NNNNN B NNNNN Perform reverse transcription (page 19) Purify the cdna (page 20) Size select the cdna (page 21) cdna 5 PCR Primer Amplify the cdna (page 25) Purify the amplified DNA (page 27) P1 sequence RNA sequence (Barcode) optional internal adaptor (IA) 3 PCR Primer barcode (BC) P2 sequence Assess the yield and size distribution of the amplified DNA (page 28) Proceed with SOLiD System templated bead preparation Refer to the Applied Biosystems SOLiD 4 System Templated Bead Preparation Guide (PN ) P1 IA BC P2

4 RNA-seq Analysis Pipeline Done on FG Stream server Many of you now have accounts (first initial and last name, one word lowercase) Password is on the board Login through Cygwin, Terminal, ect... ssh Change password (make it strong): passwd <username>

5 Useful Commands Go up a directory (cd..) Change directory (cd <directory>) list contents with details (ls -l) Read text file (more <filename>) Make a directory -only in your home directory (mkdir) Create a symbolic link (ln -s </path/ filename>

6 Where s the data? Data is located here: /data/ List contents of this directory (ls) F3, F5, Samples_fall2011_hs.txt Look at Samples file (more Samples...) This tells you which Library is for which experiment For example: Library 25 is S. bayanus control Each sample has F3 reads and F5 reads (paired-end data)

7 Making Directories for Fall 2011 Heat Shock Data Your home directory is here: /home/ <username>/ Assignment: make directory labeled <hs_fall2011> in your home directory Make the following directories in <hs_fall2011>: <Sbayanus>, <Scerevisiae>, <Smikatae>, <Sparadoxus> Make the following directories in each of those: <control>, <experimental> Make the following directories in each of those: <F3>, <F5>

8 Link Data to Your Home Directory Your home directory is here: /home/ <username>/ You may only write files to your home directory RNA-seq data files are large Want to link them to your home directory rather than copy them For example, in your S. bayanus control F3 directory type: ln -s /data/f3/ SA12003_01_Library25_Library25_F3.csfasta SA12003_01_Library25_Library25_F3.csfasta

9 What form is the data in? SOLiD sequencing data Two associated files for each F3 and F5 reads.csfasta and.qual These two files are used as input files to make a single.fastq file Use a script (program) to make a.fastq file from the linked.csfasta and.qual files in your directory

10 .csfasta.qual solid2fastq_v2.pl.fastq.fastq.fasta (reference).fastq.sai.fasta (reference) bwa aln bwa sampe.sai.sam

11 .sam.txt gene counts grep/awk Excel.txt gene counts expression data expression data convert to.csv heat map

12 Step 1: Making a.fastq file Use solid2fastq_v2.pl script (program) If unsure how to use any script, just type it in and hit enter - instructions follow If you have x.csfasta and x_qv.qual run it as: solid2fastq_v2.pl x x_out This will generate the output: x_out.single.fastq example in S. bayanus control F3 : solid2fastq_v2.pl SA12003_01_Library25_Library25_F3 Sb_c_F3

13 Aside: Tips on Running Scripts Use TAB for fill in function (saves on typing) Write nohup at beginning of command - stands for no hangup - won t interrupt if you disconnect from server When using nohup, errors usually printed to screen are printed to text file: nohup.out Type & at end of command to place it in a queue and allow you to keep working example in S. bayanus control F3 : nohup solid2fastq_v2.pl SA12003_01_Library25_Library25_F3 Sb_c_F3 &

14 Step 2: Using bwa align GSAF Wiki good resource for BWA commands (and other software) display/gsaf/bwa We are working with colorspace data (initially a.csfasta file) - need to use -c flag Using.fastq file made from solid2fastq_v2.pl Aligning to reference genome (located in / saccharomyces_refs/) - A list of ORF IDs Use Sbay_extended-2.fasta, Scer_extended-2.fasta, Smik_extended-2.fasta, Spar_extended-2.fasta

15 Step 2: Using bwa align Run it as: bwa aln -c <reference.fasta> <experiment name>.fastq > out.sai This will generate the output: out.sai If you want, you can print the screen messages to a text file as a log Do this by adding 2> out.log example in S. bayanus control F3 : nohup bwa aln -c /saccharomyces_refs/ Sbay_extended-2.fasta Sb_c_F3.fastq > Sb_c_F3.sai 2> Sb_c_F3_sai.log &

16 Step 3: Using bwa sampe Paired-end alignment Using.fastq file made from solid2fastq_v2.pl and.sai file made from bwa aln Uses.sai and.fastq inputs from both F3 and F5 reads Aligning to reference genome

17 Step 3: Using bwa sampe Run it as: bwa sampe <reference.fasta> F3.sai F5.sai F3.fastq F5.fastq > out.sam This will generate the output: out.sam Example: nohup bwa sampe / saccharomyces_refs/sbay_extended-2.fasta Sb_c_F3.sai Sb_c_F5.sai Sb_c_F3.fastq Sb_c_ F5.fastq > Sb_c.sam & This will generate the output: Sb_c.sam

18 Step 3: Using bwa sampe This.sam file will contain ALL of the reads, whether they align to the reference or not - to filter add awk '{if (substr($1,1,1)=="@") {print $0} else {if ($3!="*") {print $0}}}' With filter: nohup bwa sampe / saccharomyces_refs/sbay_extended-2.fasta Sb_c_F3.sai Sb_c_F5.sai Sb_c_F3.fastq Sb_c_ F5.fastq awk '{if (substr($1,1,1)=="@") {print $0} else {if ($3!="*") {print $0}}}' > Sb_c.sam &

19 Step 4: Counting Genes The.sam file gives the ORF ID that each pair of sequences aligned to We can list these ORF IDs and count the number of times each comes up Will use an awk and grep commands awk and grep are tools that manipulate text files

20 Step 4: Counting Genes grep -v <file>.sam awk '{print $3}' sort uniq -c awk '{print $2 "\t" $1}' > out.txt example: grep -v Sb_c.sam awk '{print $3}' sort uniq -c awk '{print $2 "\t" $1}' > Sb_c_count.txt

21 Step 5: Manipulate Gene Counts in Excel Open gene count text file in excel Find the total of all counts In another column, divide each gene s counts by total (normalization) Find log2 ratio of normalized experimental / normalized control