Subsampling a fastq file with awk

What you are going to find here

  1. A minimal introduction of the awk command in Linux and Mac (For Mac user, installing GNU awk might be necessary. It introduced some new functions like sorting an array with asort().)
  2. An awk command that would randomly subsample k reads from a given fastq file of a pair-ended sequencing.

Why I am making this note

In single cell RNA-sequencing, there seems to be no good way telling how deep you should sequence to date. Required sequencing depth should adequately capture library complexity, which is largely determined by the technique used to collect and barcode the single cell sample, and practically, we only need as much information as we need to uncover the underlying heterogeneity in the sample. Some people went very far to a minimum of 5,000,000 reads per cell, while some people suggested a much humbler number of 300,000 reads minimum per cell should suffice. With this huge variation, it might be best to decide empirically which fits your need.

Before I could actually try that, I realized I haven’t really seen R as a programming language. That is, I feel comfortable with doing analysis in R but think little about how it is computing my stuffs. This time, I finally realized I don’t know how to ask R to process a file sequentially when I tried to subsample a fastq file that was larger than my RAM.

I googled, and awk jumped as a somewhat popular answer. I quickly decided to do this subsampling with it for two reasons:

  1. awk commands look quite short.
  2. People I know are good in analysis use awk well, and I want to be like them. (Yeah, I know it’s just an illusion that I would become like them just by merely learning awk, but learning new things is alway fulfilling.)

It turned out awk is quite neat, so I decided to take a quite note here. Many of this note is adapted from the official guide of gawk, the GNU implementation of awk.

First, I’ll pipe the results of ls -l for awk to process. The goal here is to print a user-defined variable k, and then print every entry of the result of ls -l as long as its owner is root.

A minimal awk command for me to get acquaintedwith

# Command for easy copy and paste
ls -l | awk -v k=3 'BEGIN{print k} $3 == "root" {print $0} END{}'

Just like any other command in *nix system, awk takes options right after it. -v declares a variable here, and I need this because I’ll use this option to set the goal number of reads yielded from subsampling.

Then, the program part of awk is enclosed by a pair of single quote, and inside the quote, every part is comprised of a pattern and an action, which would be executed if the pattern is matched.

BEGIN is a special rule in awk pattern, the code enclosed by brackets after it would only be run once before the file begin being processed. As a result, the example command would first print k, which is 3, before everything.

Fields in awk are referred toas $N

When awk reads a file, it automatically separates every row of the file into fields. A field is pretty similar to the idea of a column of a data frame in R. The thing separating the fields could be set with option -F (awk -F "," ... sets the field separator as a comma) or simply assign it to built-in variable FS (awk 'BEGIN{FS=","}...' also sets the field separator as a comma).

Now, the short command becomes more readable: we set a variable k as 3, and then before processing the data from ls -l, print k first. Then, if field 3 ($3) is “root”, print that row for us.

Subsampling a fastq file

When I was googling, I found a kind tutorial on how to do this. Thanks to it, I got a thing to start from and watch in awe how smart reservoir sampling is.

The original command from the tutorial is:

paste forward.fastq reverse.fastq | awk '{ printf("%s",$0); n++; if(n%4==0) { printf("\n");} else { printf("\t");} }' |
awk -v k=10000 'BEGIN{srand(systime() + PROCINFO["pid"]);}{s=x++<k?x- 1:int(rand()*x);if(s<k)R[s]=$0}END{for(i in R)print R[i]}' |
awk -F"\t" '{print $1"\n"$3"\n"$5"\n"$7 > "forward_sub.fastq";print $2"\n"$4"\n"$6"\n"$8 > "reverse_sub.fastq"}'

This command will:

  1. paste the fastq to keep the paired reads together.
  2. Use awk to concatenate every 4 lines in one line, separated by tab (Because in a fastq file, a read is comprised of 4 lines).
  3. Let’s say I want only 10,000 reads: I set k as 10,000. In the BEGIN block, and I set random seed with srand(). Then, it’s show time for reservoir sampling: I take the first k-1 row, and then for every coming row, the nth row has a chance of k/n of being selected and stored in an array R.
  4. Because we concatenated first the two files with paste and then awk in step 2, we now use awk again to extract every odd field (forward read) and even field (reverse read) separately, and redirect them into forward_sub.fastq and reverse_sub.fastq respectively.

This script troubled me a bit because it sometimes draws k samples and other times only k-1 samples, so I ended up checking the definition of the sampling method and felt it is more appropriate to take the first k row instead of k-1 row in step 3.

Another challenge I encountered is that, in the command above, the selected k reads were stored in an array until the end of the program. If k reads already outsize your RAM, the command failed somewhere when there was no longer any space for the array to grow (with an odd error message killed).

My solution here is to draw sample to generate a list of picked lines first, and then for every line, we check if it is picked. If it is, we write it to a file. After that, we do aforementioned step 4 to extract the fields into sepearate fastq files.

paste forward.fastq reverse.fastq |\
awk '{ printf("%s",$0); n++; if(n%4==0) { printf("\n");} else { printf("\t");} }' |\

# Pick random sample from line numbers first
awk -v k=423549353 'BEGIN{ srand(systime() + PROCINFO["pid"]);\
for(i = 1; i <=847098706 ; i++) { s= i<=k?i-1:int(rand()*i);\
if(s<k) R[s]=i}; n=asort(R)}\

# Check the line numbers
{if(NR==1) co=1; if(NR == R[co]) {print $0 >> "temp.fastq";co++}'

# Reformat the subsampled file
awk -F"\t" '{print $1"\n"$3"\n"$5"\n"$7 > "forward_sub.fastq";\print $2"\n"$4"\n"$6"\n"$8 > "reverse_sub.fastq"}' temp.fastq
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