Sequence Length Distribution From A Fastq File

Question

[deepthithomaskannan]

Hi all,

Any script available for doing sequence length disrtibution of fastq file?

Thanks, Deepthi

[oars]

Hello,

I am trying to do something similar, were I want to determine both the read length and how many reads are in my fastq file. Here is my code:

gunzip -c SRR1060507_1.fastq.gz|awk 'NR%4==2{printlength($0)}'|uniq -c

But I keep getting the following error:

awk: cmd. line:1: (FILENAME=- FNR=2) fatal: function `printlength' not defined

I'm not sure what I've done incorrectly? I also tried Frederic's code above and although I got that to run, its not exactly the output I'm seeking, I should be returning something like 2420797 100

Any help would be super appreciated!

[michael.ante]

Between print and length should be at least one space. If you want to compute the number of reads simultaneously, use something like Frèdèric's approach

awk 'NR%4 == 2 {lengths[length($0)]++ ; counter++} END {for (l in lengths) {print l, lengths[l]}; print "total reads: " counter}' file.fastq

[oars]

Thanks! This was a winner.

Answer 1

[Frédéric Mahé]

Here is a solution using awk:

awk 'NR%4 == 2 {lengths[length($0)]++} END {for (l in lengths) {print l, lengths[l]}}' file.fastq

It reads like this: every second line in every group of 4 lines (the sequence line), measure the length of the sequence and increment the array cell corresponding to that length. When all lines have been read, loop over the array to print its content. In awk, arrays and array cells are initialized when they are called for the first time, no need to initialize them before.

[Manu Prestat]

Thanks for teaching me the NR%4 line grouping method: pretty useful indeed when dealing with fastq files.

Answer 2

[bastienllamas]

Another awk solution, very similar to Frédéric's:

cat reads.fastq | awk '{if(NR%4==2) print length($1)}' | sort -n | uniq -c > read_length.txt

And to quickly obtain a graph in R:

reads<-read.csv(file="read_length.txt", sep="", header=FALSE)
plot (reads$V2,reads$V1,type="l",xlab="read length",ylab="occurences",col="blue")

[PoGibas]

no need to use cat

Answer 3

[Jordan]

You can use FASTQC. It gives you all the details you need, including the quality, overrepresented sequences, enriched k-mers etc.

[lovegenetics]

Can you use the FASTQC on nanopore reads?

[GenoMax]

You could do that but you may want to use NanoPlot instead. It will generate stats that are specific for nanopore sequencing and thus more useful.

Answer 4

[Brian Bushnell]

Another possibility, from BBMap:

readlength.sh in=reads.fq out=histogram.txt

The default is 10bp bins with a max of 80kbp, but those can be configured (run the shellscript with no arguments for details). It's very fast, and handles fastq/fasta/sam/bam; raw/gzip/bzip2.

[al-ash]

Really useful! Only if I want to parse the output (e.g. pipe it into grep) I have to first suppress the stderr message: Processed XY reads. Time: Z seconds. Might it be more convenient to have this info also in stdout?

Answer 5

[Buffo]

Save it as fastq_lenght.py and run it :)

#!/usr/bin/env python
#-*- coding: UTF-8 -*-
import sys
######################################################################################
syntax = '''
------------------------------------------------------------------------------------
Usage: python fastq_lenght.py file.fastq 
result: .txt file same name as input name plus "_lenghts.txt" 
------------------------------------------------------------------------------------
'''
######################################################################################

if len(sys.argv) != 2:
    print syntax
    sys.exit()

######################################################################################

fastq_file = open(sys.argv[1], 'r')
prefix = sys.argv[1].split('.')[0]
outfile = open(prefix + '_' + 'lenghts.txt', 'w')
seq = ''
for line in fastq_file:
    line = line.rstrip('\n')
    if line.startswith('@'):
        if seq:
            outfile.write(name + '\t' + str(len(seq)) + '\n')
            seq = ""
        name = line 
    else:
        seq = line 
outfile.write(name + '\t' + str(len(seq)) + '\n')
fastq_file.close()
outfile.close()

print '\n' + '\t' + 'File: ' + prefix + '_' + 'lenghts.txt has been created...'

Answer 6

[Manu Prestat]

You can do this using fastx toolkit, especially, you might be interested in this program:

fastx_nucleotide_distribution_graph.sh

and maybe:

fastx_quality_stats
fastq_quality_boxplot_graph.sh

[Malcolm.Cook]

i think this does not provide length distribution information

Answer 7

[Ashutosh Pandey]

Use this post as a help Fastq Quality Read and Score Length Check and apply a few basic unix commands like sort and uniq to get the frequency.