Overview

As mentioned in the introduction tutorial as well as the read processing tutorial, read processing can make a huge impact on downstream work. While cutadapt which was introduced in the read processing tutorial is great for quick evaluation or dealing with a single bad sample, it is not as robust as some other trimmers in particular when it comes to removing sequence that you know shouldn't be present but may exist in odd orientations (such as adapter sequences from the library preparation). This tutorial is adapted from the 2021 trimmomatic tutorial which sought to do the same basic things as fastp: get rid of adapter sequences first and foremost, ideally even before fastQC so you can make any quality or length based improvements on actual data not artifacts. The #1 biggest reason why fastp is now the instructor's preferred trimming program is this box taken from the trimmomatic tutorial:

The more collaborative your work is, the less confidence you will have in picking the correct adapter file with trimmoatic, and while thanks to conda installations it can be pretty easy to test multiple different ones, fastp does all the guess work for you, and can generate some interesting graphs itself.

Learning objectives:

1. Install fastp

2. Remove adapter sequences from some plasmids and evaluate effect on read quality, or assembly.

Installing fastp

fastp's home page can be found on github and has links to the paper discussing the program, installation instructions for conda, and information on each of the different options available to the program. This is far above the quality the average programs will have as most will not have a user manual (or not nearly so detailed), may not have been updated since originally published (or may not have been published), etc. It having been updated since the publication is one thing that makes it such a good tool as the more who use it the more likely problems are found, and having a group who is going to actively improve the program will significantly increase its longevity.

conda create -n GVA-ReadPreProcessing -c bioconda -c conda-forge fastp fastqc multiqc

Trimming adapter sequences

Example generic command

fastp -i <READ1> -I <READ2> -o <TRIM1> -O <TRIM2> --threads # --detect_adapter_for_pe -j <LOG.json> -h <LOG.html>

Breaking down the parts of the above command:

-j <LOG.json>

-h <LOG.html>

All of the above has been put together from the help fastp --help command.

Trimming a single sample

Get some data

mkdir -p $SCRATCH/GVA_fastp_1sample/Trim_Reads $SCRATCH/GVA_fastp_1sample/Raw_Reads
cd $SCRATCH/GVA_fastp_1sample
cp $BI/gva_course/plasmid_qc/E1-7* Raw_Reads

The ls command should show you 2 gzipped fastq files. You may notice that here that we used a wildcard in the middle of our copy path for the first time. This is done so that you can grab both R1 and R2 easily without having to type out the full command. Double tab will help tell you when you have a sufficiently specific base name to only get the files you are after.

Trim the fastq files

The following command can be run on the head node. Like with FastQC if we are dealing with less than say 1-2Million reads, it is reasonable to run the command on the head node unless we have 100s of samples in which case submitting to the queue will be faster as the files can be trimmed all at once rather than 1 at a time. Use what you have learned in the class to determine if you think this command should be run on the head node. (this was covered in more detail in the first part of the evaluating and processing read quality tutorial.)

zgrep -c "^+$" Raw_Reads/*.fastq.gz

fastp -i Raw_Reads/E1-7_S187_L001_R1_001.fastq.gz -I Raw_Reads/E1-7_S187_L001_R2_001.fastq.gz -o Trim_Reads/E1-7_S187_L001_R1_001.trim.fastq.gz -O Trim_Reads/E1-7_S187_L001_R2_001.trim.fastq.gz -w 4 --detect_adapter_for_pe 

Evaluating the output

Using everything you have learned so far in the class, can you answer the following questions?

1. 
   

   E1-7_S187_L001_R2_001.trim.fastq.gz and E1-7_S187_L001_R1_001.trim.fastq.gz These were created with the -o and -O options, they are in the Trim_Reads folder, and you likely found them using the ls command fastp.html and fastp.json These are log files created by default since we didn't specify their names. This is part of why -j and -h were discussed above with the general command.  While the json file can be evaluated in the terminal (cat less more head tail), the html file has to be transferred back to your computer to view.

   
   

2. 
   

   5884 paired end reads 11768 total reads You likely found this out from using the zgrep command, or from the following blocks that printed as the command ran: Read1 after filtering: total reads: 5884 total bases: 791763 Q20 bases: 782948(98.8867%) Q30 bases: 765510(96.6842%) Read2 after filtering: total reads: 5884 total bases: 791763 Q20 bases: 711414(89.8519%) Q30 bases: 658164(83.1264%) Filtering result: reads passed filter: 11768 reads failed due to low quality: 2014 reads failed due to too many N: 0 reads failed due to too short: 0 reads with adapter trimmed: 3970 bases trimmed due to adapters: 193972

   
   

3. 
   

   From the information generated while the command ran we see: Insert size peak (evaluated by paired-end reads): 171 This tells us that the average peak size was 171 bases, and that it was estimated by looking at the overlap between the read pairs. It is potentially inaccurate as reads which do not overlap each other can not estimate the size. If you transferred the .html file back to your laptop, you would see this relevant histogram: The general section of the summary at the top of the html tells us that the average insert size was 171, while the histogram tells us that 50% of our data is <18 or >272 bases

   
   

4. 
   

   If you only look at the information that printed to the screen, you probably answer "No" you likely see the following block and think this is the end of the answer: Detecting adapter sequence for read1... No adapter detected for read1 Detecting adapter sequence for read2... No adapter detected for read2 A more fuller answer might be "maybe" or "probably" or "I'm not sure" as: 1. Not finding any adapter would be super rare 2. If 45% of our reads have an insert size of 171 bases, and we did 151bp PE sequencing,  we should be able to find adpater sequences 3. in the filtering results we see: Filtering result: reads passed filter: 11768 reads failed due to low quality: 2014 reads failed due to too many N: 0 reads failed due to too short: 0 reads with adapter trimmed: 3970 bases trimmed due to adapters: 193972 If you look at the html file you probably answered "yes" There is a section for Read1 and Read2 adapters which show a growing stretch of DNA which recreates the illumina adapter sequences.

   
   

5. 
   

   Like we saw in our fastqc reports (over represented sequences having "no hit" and adapter content staying at bottom of graph), for something to be classified as an "adapter" in the first section of the printed information, it has to meet certain criteria that in this (and many other instances) is perhaps a bit too stringent.

   
   

6. 
   

   This is pretty open ended, take a look at the html file in particular, see what of it does or doesn't make sense and consider asking a question if you would like to know more.

   
   

Trim all the samples from the multiqc tutorial

As mentioned above, if you have already done the multiqc tutorial, you can use your new fastp command to remove the adapter sequences from all 544 samples or make other changes based on what you saw from the fastqc/multiqc reports. 

Get some data

mkdir -p $SCRATCH/GVA_fastp_multiqcSamples/Trim_Reads
cd $SCRATCH/GVA_fastp_multiqcSamples
cp -r $BI/gva_course/plasmid_qc/ Raw_Reads/

 The ls command will now show 2 directories, and like in the multiqc tutorial, you can use ls and wc commands to figure out how many files you will be working with.

Trim the fastq files

Just as we used a for loop to set up a set of FastQC commands in the multiqc tutorial, we can use a similar for loop to generate a single file with 272 trim commands for the 544 total files.

The following command will pair all R1 reads in the Raw_Reads folder with its R2 pair, determine the base name, and generate a command to trim the file 

for r1 in Raw_Reads/*_R1_*.fastq.gz; do r2=$(echo $r1|sed 's/_R1_/_R2_/'); name=$(echo $r1|sed 's/_R1_001.fastq.gz//'|sed 's/Raw_Reads\///'); echo "fastp PE $r1 $r2 -baseout Trim_Reads/$name.fastq.gz ILLUMINACLIP:TruSeq3-PE-2.fa:4:30:10 MINLEN:30";done > trim_commands

for R1 in Raw_Reads/*_R1_001.fastq.gz; do R2=$(echo $R1| sed 's/_R1_/_R2_/'); name=$(echo $R1|sed 's/_R1_001.fastq.gz//'|sed 's/Raw_Reads\///'); echo "fastp -i $R1 -I $R2 -o Trim_Reads/$name.trim.R1.fastq.gz -O Trim_Reads/$name.trim.R2.fastq.gz -w 4 --detect_adapter_for_pe -j 04_Trim_Logs/$name.json -h 04_Trim_Logs/$name.html &> 04_Trim_Logs/$name.log.txt";done > fastp.commands

Use the head and wc -l to see what the output is and how much there is of it respectively.

Again as we discussed in the multiqc tutorial, running this number of commands is kind of a boarder line case, there are not a lot of total reads, but there are a large number of samples and our command does request additional processors so we should not be on the head node. 

cp /corral-repl/utexas/BioITeam/gva_course/GVA2022.launcher.slurm trim.slurm
nano trim.slurm

Again while in nano you will edit most of the same lines you edited in the in the breseq tutorial. Note that most of these lines have additional text to the right of the line. This commented text is present to help remind you what goes on each line, leaving it alone will not hurt anything, removing it may make it more difficult for you to remember what the purpose of the line is

The changes to lines 22 and 23 are optional but will give you an idea of what types of email you could expect from TACC if you choose to use these options. Just be sure to pay attention to these 2 lines starting with a single # symbol after editing them.

Again use ctl-o and ctl-x to save the file and exit.

sbatch trim.slurm

The job should take less than 10 minutes once it starts if everything is working correctly, the showq -u command can be used to check for the job finishing.

Evaluating the output

ls Trim_Reads | wc -l
grep -c "TrimmomaticPE: Completed successfully" Queue_job.o*
grep -c "100.00%" Queue_job.o*

The above 3 commands are expected to show 1088 and 272 and 0 respectively, if you see other answers it suggests that something went wrong with the trimming command itself. If so remember I'm on zoom if you need help looking at whats going on. The most common error that I expect will be that you ran out of ram by trying to process too many samples at once (such as if you used a -n 68 option in your .slurm file). 

Beyond the job finishing successfully, the best way to evaluate this data would actually be to move back to the multiqc tutorial and repeat the analysis there that was done on the raw files for the trimmed files here.

Personal experience

The for loop above focuses just on generating the trim commands. In my experience that is only half of the job, the other half is capturing the individual outputs so you can evaluate how many reads were trimmed in what way for each sample. Perhaps you will find this command helpful in your own work: 

mkdir trimLogs; mkdir Trim_Reads; for r1 in Raw_Reads/*_R1_*.fastq.gz; do r2=$(echo $r1|sed 's/_R1_/_R2_/'); name=$(echo $r1|sed 's/_R1_001.fastq.gz//'|sed 's/Raw_Reads\///'); echo "trimmomatic PE $r1 $r2 -baseout Trim_Reads/$name.fastq.gz ILLUMINACLIP:TruSeq3-PE-2.fa:4:30:10 MINLEN:30 >& trimLogs/$name.log"; done > trim_commands

After the job completes, the following command is useful for evaluating its success:

echo -e "\nTotal read pairs:";wc -l trim_commands;echo "Successful trimmings:"; tail -n 1 trimLogs/*|grep -c "TrimmomaticPE: Completed successfully";echo "Potential trimming errors:"; cat trimLogs/*|grep -c "100.00%"

This gives me a quick readout of if all of the individual commands finished correctly.

Further, if you were to use this for loop rather than the one listed in the tutorial above, you would see each sample generates its own log file in the the trimLogs directory that when investigated with cat/more/less/tail/nano is identical to the output you saw when you trimmed a single sentence allowing you to figure out how different samples are being processed.

Optional next steps:

1. Consider moving back over to the multiqc tutorial use multiqc to determine well the trimming worked. 
2. The reads could then further be assembled in the Spades tutorial as they are all plasmid samples.

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