Mapping _ unmapped reads

[GGOPA]

Hi,
For the RNA-Seq analysis( paired-end stranded libraries for cell cultures infected with virus) of one of my samples (triplicate)I see only around 40% over mapping (HISAT2) to host genome. I tried using RNASTAR and the resut was similar. I wanted to know if all the unmapped reads belong to the viral genes, if it is not from the host.Therefore, I chose the option to write the unaligned reads separately while re-running HISAT2. I got 2 files’HISAT2 on data:unalined reads (L)’ and ‘HISAT2 on data:unlined reads (R)’.

Q1. What is L and R here? Do they denote pairs, as my library was paired-ends? If so, which one should I choose as my Forward read and Reverse read when I do the mapping?

Q2. Can you please suggest an appropriate method to see what proportion of these unmapped reads belonged to the virus?

Many thanks,G

[gallardoalba]

@GGOPA

Q1. What is L and R here? Do they denote pairs, as my library was paired-ends? If so, which one should I choose as my Forward read and Reverse read when I do the mapping?

Q2. Can you please suggest an appropriate method to see what proportion of these unmapped reads belonged to the virus?

Hi @GGOPA,
the L corresponds to forward reads and R with the reverse ones. In order to analyze the origin of the reads, I suggest you to use Kraken2 in order to perform a taxonomical classification of the reads; you can find some information about it in this training.

Regards