I am new to processing and analysis of RNAseq data. I have recently completed a paired-end alignment using mm10 as a reference genome. I selected to "Specify strand-specific information as FR Unstranded" and "Disable spliced alignment as no-spliced-alignment". Most of the other settings I left as default. The following alignment was relatively poor:
79081475 reads; of these: 79081475 (100.00%) were paired; of these: 47291164 (59.80%) aligned concordantly 0 times 26796680 (33.88%) aligned concordantly exactly 1 time 4993631 (6.31%) aligned concordantly >1 times ---- 47291164 pairs aligned concordantly 0 times; of these: 1181324 (2.50%) aligned discordantly 1 time ---- 46109840 pairs aligned 0 times concordantly or discordantly; of these: 92219680 mates make up the pairs; of these: 55187078 (59.84%) aligned 0 times 30703132 (33.29%) aligned exactly 1 time 6329470 (6.86%) aligned >1 times 65.11% overall alignment rate
I tried trimming prior to alignment as the FastQC of the reverse showed poor per base sequence content however the alignment results were similar.
I would be very grateful for any help on how to improve the alignment. Thank you very much for your time.