I'm very new to sequencing analysis and I'm struggling to understand the differences between the above reads and ways to find them. I have a ChIP-seq paired-end dataset and I want to get the above statistics for publication.
Raw reads: Is this the total reads present in the fastq file? So a paired-end sequencing has two files which means I'll add the total number of reads from both the files to get this number?
Clean reads: Is this the number of reads I get after the quality control step? Is this also the combination of both the files?
total mapped reads: I believe this as all the reads mapped to my genome.
unique mapped reads: ?
I also use Picard tool to remove duplicates and in the metrics, I found that it has unpaired read duplicates and paired read duplicates, how can I get a single number from this as the number of duplicates? Is it just the addition of two or do I need to double the pair duplicate count and add it with the unpaired count?
Also, for publishing ChiP-seq data what are all the essential statistics including/apart from the above?
Thank you.
1 answer
Raw reads: Is this the total reads present in the fastq file? So a paired-end sequencing has two files which means I'll add the total number of reads from both the files to get this number?
Correct. Each cluster (in Illumina) produces those two reads from two ends of each library fragment. Paired end reads are in separate files so you don't need to add those numbers. Each unique cluster produces two reads in paired-end sequencing.
Clean reads: Is this the number of reads I get after the quality control step? Is this also the combination of both the files?
Paired-end read files are qc'ed and scanned/trimmed together so the read number in both files should remain identical post-processing.
total mapped reads: I believe this as all the reads mapped to my genome.
Some reads may have secondary/supplementary alignments (multi-mapping) so the number may look larger than actual number of reads.
unique mapped reads: ?
Like what it says. Reads mapped one time in genome as pair.
Also, for publishing ChiP-seq data what are all the essential statistics including/apart from the above?
Probably nothing in the list above would need to go in a publication. This is part of standard analysis.
With ChIPseq you expect to see a lot of duplicate reads since you are enriching for regions in the genome.
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