1. Setting up visualization

2. Make output folder for visualization files Log into vieques $ ssh amso5880@vieques.colorado.eduamso5880@vieques.colorado.edu Change directories $ cd /projects/sreadgroup/students $ pwd $ ls Make output folder $ mkdir $ ls

3. Follow directions for setting up your vnc session Check email from Jamie Kershner for port and link to setup vnc session http://biof-edu.colorado.edu/videos/dowell- short-read-class/visualization-information

4. What do I do when I get data back from the sequencer?

6. Prep your home directory $ cd /Users/identikey/ $ mkdir Day4 $ cd Day4 $ mkdir fastq $ mkdir $ mkdir PBS $ mkdir PBS/eofiles/

7. Copy today’s files $ scp /projects/sreadgroup/Day4/fastq/ * /Users/identikey/Day4/fastq $ cp /projects/sreadgroup/Day4/PBS/* /Users/identikey/Day4/PBS

8. Unzip your files $ cd /Users/identikey/Day4/fastq $ ls $ gunzip * $ ls $ less Example_A_01.fastq

9. What is a Fastq file? Read Identifier Sequenc e Quality

10. For the remainder of the FastQC examples, the class will be split into 4 groups Group A – Work with Example_A files Group B – Work with Example_B files Group C – Work with Example_C files Group D – Work with Example_D files DO NOT DELETE the other files from your folder, they will be used in your homework

11. Concatenate fastq files $ wc –l Example_X_01.fastq $ wc –l Example_X_02.fastq $ cat Example_X_0*.fastq > Example_X.fastq $ ls -lh $ wc –l Example_X.fastq $ rm Example_X_0*.fastq

12. Evaluating Sequencing and Library Quality

13. Where to find FastQC options $ /opt/fastQC/fastqc --help -OR- $ module load fastqc_0.11.2 $ fastqc --help Don’t know how to use a program? Look at the help page for options!

14. Running FastQC $ cd /Users/identikey/Day4/PBS/ $ ls $ nano run_fastqc.pbs

15. Specify Job name Update resources if necessary Specify Queue Update eo file path #PBS –e /Users/identikey/Day4/PBS/eofiles/ #PBS –o /Users/identikey/Day4/PBS/eofiles/ Update email Specify output dir -o /Users/identikey/Day4/QC/ Specify input file /Users/identikey/Day4/Example_X.fastq Replace anything with " " with your desired value & "amso5880" with your identikey

16. Submit FastQC job $ module list $ module load fastqc_0.11.2 $ qsub run_fastqc.pbs $ qstat –u identikey

17. Look at eofiles $ cd eofiles $ ls $ less jobname.e12345 $ less jobname.o12345

18. Locate output files $ cd /Users/identikey/Day4/QC $ ls Move html file to visualizer folder $ cp Example_X_fastqc.html / projects/sreadgroup/students/ identikey/ OR: Use FileZilla to transfer html files to your own computer

20. Running Trimmomatic: Quick Start Settings $ java –jar /opt/trimmomatic/ 0.32/trimmomatic-0.32.jar $ cd /Users/identikey/PBS $ nano QS_trim.pbs

21. Job name Queue eofiles Email Replace anything with " " with your desired value and "amso5880" with your identikey

22. Trimmomatic Settings java -jar /opt/trimmomatic/0.32/trimmomati c-0.32.jar SE -threads 4 - phred33 call program Single end setting Multithreading Phred33 quality scores

23. Trimmomatic Settings Trimlog -trimlog /Users/identikey/Day4/ fastq/input_fastq.trimlog Input file /Users/identikey/Day4/fastq/input.f astq Output file /Users/identikey/Day4/fastq/input_t rimmed.fastq

24. Trimmomatic Settings ILLUMINACLIP:/opt/trimmomatic/0. 32/adaptors/TruSeq3-PE- 2.fa:2:30:10 \ LEADING:3 \ TRAILING:3 \ SLIDINGWINDOW:4:15 \ MINLEN:36

25. Run trimmomatic & look at eofiles $ qsub QS_trim.pbs $ qstat –u identikey $ cd eofiles $ ls $ less job_name.e12345 $ less job_name.o12345

26. Check trimlog and fastq_trimmed files $ cd /Users/identikey/Day4/fastq $ ls $ head -12 input.fastq $ head -12 input_trimmed.fastq $ less input_fastq.trimlog

27. Copy trimmomatic PBS to a new file $ cd../PBS $ ls $ cp QS_trim.pbs new_trim.pbs $ nano new_trim.pbs

28. Update new_trim.pbs Job name Settings java -jar /opt/trimmomatic/0.32/trimmomatic- 0.32.jar SE -threads 4 -phred33 \ -trimlog /Users/amso5880/Day4/fastq/ input_fastq_cropped.trimlog \ input.fastq\ input_cropped.fastq \ CROP:40 \ HEADCROP:10

29. Run trimmomatic & look at eofiles $ qsub new_trim.pbs $ qstat –u identikey $ cd eofiles $ ls $ less new_job_name.e12345 $ less new_job_name.o12345

30. Check trimlog and fastq_trimmed files $ cd /Users/identikey/Day4/fastq $ ls $ head -12 input.fastq $ head -12 input_cropped.fastq $ less input_fastq_cropped.trimlog