Is the RNA- sequencing quality too bad for analysis?

Question

[Saran]

Hello,

I have two PCR amplicons that have been multiplexed and sequenced. I have PCR-amplified Paired-End Illumina reads (2x150) and have attempted to align them to a fasta file with both of the amplicon sequences which are 222 and 228 bp. I attempted to use both minimap2 and bwa-mem.

bwa-mem output the following:

[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] skip orientation FR as there are not enough pairs
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs

with the following lines in the sam file (none were mapped):

MN01173:264:000H57WYJ:1:11101:5825:1126 77  *   0   0   *   *   0   0   GGCGGCCCCTTGTTTGATGTNCAGAGGGAGNTGAGATCGGAAGAGCACACGTCTGAANTCNAGNCACAGCTCACGTATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAANGGGGGGGNGGNGGGGNGGGGGGGGGGGGGGGGGGGGGGGG

I notice a large "AAAAGGGGGG" repeat region on the tail of many reads. Could this be from sequencing through adapters into the flow cell? I expect overlap but The target regions are 200+ bp so I did not expect short inserts.

I also must say that the sequencing depth was off fr many samples as sizes ranged from bytes to MB.

I ran FASTQC and I get the following results concerning adapter content:

I do not know if these reads are even usable. I have also tried merging them and I get low percentages of reads paired-ends that merge (30% max).

I am trying to figure out how many reads align to the first amplicon versus the second and I was going to use samtools idxstats for that but I am not sure this sequencing is sufficient for this analysis. If someone could let me know what else to analyze or if i could try to filter out any useful reads that would be great.

Thank You, Sara

[GenoMax]

This data does look iffy. Having 70% of your reads be less than 50 bp does not bode well in terms of you being able to distinguish between the two amplicons. You could try to merge the reads using bbmerge.sh. Then trim them to remove adapters and then align to the amplicons. Likely not going to be pretty but worth a try.

BTW: How is this related to RNA?

[LChart]

What does the BioAnalyzer run look like for the library (if one was run for it)? This looks like the library is basically full of adapter dimers (i.e., the experiment just failed).

Could this be from sequencing through adapters into the flow cell?

Yes; particularly if the only thing you are sequencing (as it looks like in this case) is adapters...