RNAseq was performed using Control and Knock down cells for a specific gene. Knock down was carried out using siRNA and samples were checked with Real time and semi quantitative PCR for knock down and it was found to be gene specific knock down only. Sequencing was performed using Illumina HiSeq platform. The data generated, passed all the QC parameters. We got 8GB data with 100 bp average read length. Q30 >96% and phred score was also greater than 38. Adaptor trimming and contamination removal was performed followed by alignment with the reference genome. Percentage of alignment with the control cells was approx 90 but the percentage alignment with Knock down remained 45 only. Alignment was performed using STAR (2.4.1d). Can any one help me with the reason why alignment was so low with the knock down?
Hi Sujit.. initially one would say that the percentage alignment means there arent enough reads specific to the reference genome (in your knock down sample).. but this is a rare scenario. So it is early to comment with the given scenario.
Could you please try aligning the same two samples with Bowtie? with and without preprocessing? Please use Qualimap for Alignment metrics.. We could see what's happening in your data.
Hello, I have done alignment with both Bowtie- TopHat and EdgeR to my Knock down samples and both gave me great percentage of alignment. You can also Deseq2.
Hello Sujit,
Considering the details you just gave, there is something wrong for sure! Perhaps you can try to analyse all the reads that are not being mapped. I would recommend you to use them in a de novo assembly, and then selecting a couple of representative contigs for a BLAST exercise. In this way, you'll be able to observe what is contributing to that high percentage of un-mapped reads.
Thank you Paulo Almeida for your reply. I am actually trying to find out the alignment of these un-mapped reads at the moment.
Thank you Rana Al-Baghdadi, we will see what we get using Deseq2.
Thank you Venkatesh Chellappa, i will work on your suggestion.
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