What Do Marked Reads Mean In An Xsq File?

Question

[Jordan]

Hi,

We have xsq files generated in our lab. And I was planning to convert them to .csfasta and .qual files for further analysis, which can be done using XSQTools. In XSQ tools we have a script called convertFromXsq.sh which does exactly that. It has a filter option -f which says that it removes marked reads. I'm not sure what marked reads are and if I should use this option.

Here is the link to the guide (page 9 has the filter option description) -> XSQ Tutorial

Can anyone tell me what it means? Thanks!

Answer 1

[Ashutosh Pandey]

I assume (this is my assumption so please don't believe it blindly) these are bad reads that didn't pass the quality criteria and were flagged as bad reads. I don't think there should be many of such reads if any present in the xsq file. I have used lifescope quiet a bit and in the end it gives you a statistics table that tells you total reads in the xsq file and how many of these got filtered before the alignment and how many of those that were not filtered got aligned and all other information. I have rarely seen any numbers in the row that mentions the numbers of reads filtered before alignment.

Answer 2

[Jordan]

I think I kinda figured out what filtering here does. Without the -f option, I would get .csfasta files with few reads like this:

>1_44_92_F3    
T..................................................

When I use -f, these kind of sequences/reads are not present in the csfasta file. But does removing these reads make any difference in the further analysis like mapping?

[Ashutosh Pandey]

It wont make any difference in terms of mapping. But in case of paired end or mate pair reads, aligners like BWA, Bowtie want that the reads belonging to a same pair ( whether paired end reads or mate pair reads) should maintain the order in fastq file. In other words, if you remove one read from the pair you should also remove the other pair irrespective of whether it is a good or bad read.

[Ashutosh Pandey]

In other words dont remove them. They wont be mapped at all or aligner will remove them for you.

[Jordan]

Makes sense. Thanks for the tip. But I use NovoalignCS for mapping. It probably follows the same logic too.