1. Stubbs Lab Bioinformatics – 4 Alignment Summary Report & Count files with htseq-count
Nov 29, 2016
Joe Troy

2. Agenda One new Linux command - htop The RNA-Seq Analysis so far
Creating an alignment summary report
Creating count files with htseq-count

3. One new Linux command - htop
htop displays a screen to monitor processes running on the stubbslab.igb.illinois.edu server.
press ‘q’ to exit the htop display
~]$ htop

4. INPUT: .tgz file(s) from ftp.biotec.illinois.edu
INPUT: .fastq short read files
INPUT: align_summary.txt files from tophat2
OUTPUT: “accepted_hits.bam” for each sample
OUTPUT: alignment_summary.txt
(can be opened in excel)
OUTPUT: .fastq short read files
OUTPUT: “align_summary.txt”
Retrieve and un-compress short read files
Align Reads to genome
Alignment Summary Report to review alignment stats
sftp command
Tophat 2 script
main_script_alignment_summary_16Gso.sh
&
create_alignment_summary.R
tar command

5. INPUT: “accepted_hits.bam” file from each sample
OUTPUT: .count files
Create Count Files
(un-normalized counts by gene)
main_script_htseq_count_16Gso.sh
note: this script uses htseq-count

6. Instructions to create an alignment summary report
INSTRUCTION SLIDE 1 of 2 – no need to use ‘screen’ as it runs quick!
Josephs-MacBook-Pro:~ josephtroy$ ssh
password:
Last login: Mon Nov 21 20:15: from c hsd1.il.comcast.net
~]$ df -h
Filesystem Size Used Avail Use% Mounted on
/dev/sda T 4.2T 150G 97% /
/dev/sda G 14G 77G 16% /var
/dev/sdb M 29M 246M 11% /boot
tmpfs G G 0% /dev/shm
/dev/sdb G 116G 145G 45% /var/lib/mysql
~]$ cd /home/share/example_rna_seq_project_16Gso/
example_rna_seq_project_16Gso]$ cd code_020_alignment_summary_report/
code_020_alignment_summary_report]$ ls -lh
total 16K
-rw-rw-r-- 1 jmtroy2 jmtroy Nov 27 12:00 alighment_report_heading.txt
-rw-rw-r-- 1 jmtroy2 jmtroy2 3.9K Nov 26 19:04 create_alignment_summary.R
-rw-rw-r-- 1 jmtroy2 jmtroy2 4.8K Nov 27 12:14 main_script_alignment_summary_16Gso.sh
code_020_alignment_summary_report]$ sh main_script_alignment_summary_16Gso.sh
[1] "start parameter loop"
[1] "end parameterloop"
end script main_script_alignment_summary_16Gso.sh at Sun Nov 27 12:20:46 CST 2016
code_020_alignment_summary_report]$

7. Instructions to create an alignment summary report
INSTRUCTION SLIDE 2 of 2
In cyberduck find the “alignment_summary.txt” file and move it (drag and drop) to a local folder on your PC or laptop.
Open the “alignment_summary.txt” file with excel on your PC or laptop.

8. Instructions to create count files with htseq-count http://www-huber
INSTRUCTION SLIDE 1 of 3
Josephs-MacBook-Pro:~ josephtroy$ ssh
password:
Last login: Mon Nov 21 20:15: from c hsd1.il.comcast.net
~]$ df -h
Filesystem Size Used Avail Use% Mounted on
/dev/sda T 4.2T 150G 97% /
/dev/sda G 14G 77G 16% /var
/dev/sdb M 29M 246M 11% /boot
tmpfs G G 0% /dev/shm
/dev/sdb G 116G 145G 45% /var/lib/mysql
example_rna_seq_project_16Gso]$ screen

9. Instructions to create count files with htseq-count http://www-huber
INSTRUCTION SLIDE 2 of 3
~]$ cd /home/share/example_rna_seq_project_16Gso/
example_rna_seq_project_16Gso]$ cd code_025_htseq_count/
code_025_htseq_count]$ ls
main_script_htseq_count_16Gso.sh
code_025_htseq_count]$ sh main_script_htseq_count_16Gso.sh …
NOW HOLD DOWN THE CONTROL KEY AND PRESS a, THEN PRESS d, TO DETACH FROM THE SCREEN SESSION
Options for monitoring a running process:
Use cyberduck (don’t forget to keep refreshing the screen) to watch the output folder.
Use the htop command to monitor system activity.
Reattach the screen session for your process. Press ENTER a few times, and when the command prompt appears the process is done.

10. Instructions to create count files with htseq-count http://www-huber
INSTRUCTION SLIDE 3 of 3 Remember: the process number ‘11559’ will be different when you run this exercise ~]$ screen -ls There is a screen on: pts-2.stubbslab (Detached) 1 Socket in /var/run/screen/S-jmtroy2. ~]$ screen -r 11559
code_025_htseq_count]$ exit
~]$ screen -ls
No Sockets found in /var/run/screen/S-jmtroy2.