Hi all,

I am working with RNA-seq data of different drug treated human cell lines sequenced on Illumina (2X150 bp chemistry). Data generated is 11-14 Gb.

I am using `HISAT2` for alignment on human genome hg38 build downloaded from [Ensembl database][1] using default parameters. To my surprise, the alignment percentage is very low (`~2 %`) for all the samples.

I have the following observations regarding the data quality :

1. Data quality (phred score) is good i.e. in the range of `32-40`!
2. Illumina adapters were already removed using trimmomatic. No other contaminants.
3. The fastqc metric '[Per base sequence content][2]' fails for all samples. More specifically, A,T,G,C % is deviating considerably at the first 10 base positions.

**Questions**

1. What could possibly be wrong? Any other QC stuff I need to re-consider?
2. More importantly, this is cell-line data, so shall I consider mapping on specific cell line reference OR is it okay if I have just the hg38 standard human reference?


  [1]: ftp://ftp.ensembl.org/pub/release-90/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.toplevel.fa.gz
  [2]: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/Help/3%20Analysis%20Modules/4%20Per%20Base%20Sequence%20Content.html
I've seen cell lines being contaminated or swapped by other lines - in the "good" cases by cell lines from other species (good because when this happens it is really easy to detect). Your low mapping rate suggests you are mapping to the wrong species, [Centrifuge][1] is really handy to detect this kind of contamination - the problem is to download or build a nt index.


  [1]: http://www.ccb.jhu.edu/software/centrifuge/