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
Hi Vijay,

Do you know how the library was prepared? Strong deviation for the first 10 bases in the nucleotide composition sounds very much like an adapter sequence. Some library preparations (eg. the [Clontech SMARTer Pico](http://www.clontech.com/CN/Products/cDNA_Synthesis_and_Library_Construction/Next_Gen_Sequencing_Kits/Total_RNA-Seq/Pico_Input_Total_RNA_Illumina_Sequencing) kit, amongst others) require trimming from the 5' of sequences before alignment. These adapters are different from the usual illumina sequenceing adapters and will be removed by default by most trimming tools.

If you know the library preparation kit, you can look up the kit's documentation and you'll usually find instructions. For example, from the [Pico docs](www.clontech.com/xxclt_ibcGetAttachment.jsp?cItemId=111761):

> IMPORTANT: The first three nucleotides of the first sequencing read (Read 1) are derived from the template-switching
oligo. These three nucleotides must be trimmed prior to mapping. 

But, as you already have the nucleotide content plot you can do without this and just remove the first 10 bases from the 5' of each read. Then your alignment scores should hopefully improve..

I hope this helps!

Phil