Visualize nucleotides for every position in R

Question

[marija]

Hi, please, how can I visualize nucelotides for every position. I have a percentage of bases but now I don't know how to create a plot.

fastq <- readDNAStringSet("https://d28rh4a8wq0iu5.cloudfront.net/ads1/data/ERR037900_1.first1000.fastq","fastq")
fastq
freq <- alphabetFrequency(fastq, as.prob = T,baseOnly=T)

I would like something like this (sorry for quality):

[Pierre Lindenbaum]

or use fastqc https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ?

Answer 1

[cpad0112]

You need to change your code. It is not alphabet frequency. It is consensus matrix. Example code and image below:

library(Biostrings)
fastq <- readDNAStringSet("sample.fastq","fastq")
## At each position, base frequency
afmc=consensusMatrix(fastq, baseOnly=T,as.prob = T)
tafmc=t(afmc)
matplot(tafmc[,-5], type="l", lwd=2, xlab="Read Length", ylab= "Base frequency at each position")
legend(legend = colnames(tafmc)[-5],"topright",col=1:4, lty=1:4, lwd=2)

5th column is others (other than A,T, G and C).

[marija]

Awesome, thank you very much.

[cpad0112]

much simpler is using SeqTools in R:

library(seqTools)
# Reads fastq file
fq=fastqq("sample.fastq")
# Plots nucleotide frequency
plotNucFreq(fq,1)

[xiaoleiusc]

Thanks, does this apply to fasta file containing sequence of one gene? How to set sliding window size in Seqtools?

Answer 2

[Sean Davis]

library(Biostrings)
fastq <- readDNAStringSet("https://d28rh4a8wq0iu5.cloudfront.net/ads1/data/ERR037900_1.first1000.fastq","fastq")
freq <- alphabetFrequency(fastq, as.prob = T,baseOnly=T)
matplot(freq,type='l')
legend(legend = colnames(freq),x=700,y=0.7,lty=1:5,col=1:5)

Note answer about consensusMatrix. That code is the best answer.

Answer 3

[chen]

This is per-cycle base content. R is not good at, and is not designed for such task.

Why complicate life? Just use fastp to do the easiest and fastest way for such tasks, including QC and filtering. It will output the figures like what you want.