Alignment >1 time represents

Question

[bioinformatics.queries]

Hello everyone

Currently I performed the alignment for DNA-seq data using Bowtie2 using hg19 as reference genome and below is what the alignment shows.

192316851 reads; of these:
  192316851 (100.00%) were unpaired; of these:
    5956226 (3.10%) aligned 0 times
    132246040 (68.76%) aligned exactly 1 time
    54114585 (28.14%) aligned >1 times
96.90% overall alignment rate

My question is are those reads aligned > 1 times should be excluded? What does > 1 time alignment stands for in sequencing? Are the alignment score obtained goo enough to be processed further. Looking ahead for response.

Thank you so much.

[bioinformatics.queries]

Thank you so much for your response. I have used DNA-seq data for alignment. Could you please tell how do you get to know its RNA-seq?

[GenoMax]

In RNAseq, RNA is converted to cDNA, which is what gets sequenced.

Could you please tell how do you get to know its RNA-seq?

You can look at the alignment of the data. Reads should predominantly align/pileup where exons/UTR are present. If you are working with bacterial data it may become tricky. In any case you will know what kind of data it is, based on metadata.

Answer 1

[GenoMax]

What does > 1 time alignment stands for in sequencing?

These are multi-mapping reads. Since reads you have are short they likely align well to multiple positions in genome (e.g. think of repeats, domains etc). These are not going to be counted (by default) by counting programs like featureCounts so will normally be excluded.

If you want to use all data then consider using a program like salmon (LINK) instead. You will need to use transcriptome with this tool.

Note: You say DNAseq but have used tag RNAseq so I assume this is RNAseq data.