[ErikFas]

Ok, here is what BBMap stdout:

  ------------------   Results   ------------------   

Genome:                	1
Key Length:            	13
Max Indel:             	16000
Minimum Score Ratio:  	0.56
Mapping Mode:         	normal
Reads Used:           	72832	(9176832 bases)

Mapping:          	2.021 seconds.
Reads/sec:       	36044.03
kBases/sec:      	4541.55


Pairing data:   	pct reads	num reads 	pct bases	   num bases

mated pairs:     	  0.1016% 	       37 	  0.1016% 	        9324
bad pairs:       	  0.0000% 	        0 	  0.0000% 	           0
insert size avg: 	  219.24


Read 1 data:      	pct reads	num reads 	pct bases	   num bases

mapped:          	  0.1181% 	       43 	  0.1181% 	        5418
unambiguous:     	  0.1181% 	       43 	  0.1181% 	        5418
ambiguous:       	  0.0000% 	        0 	  0.0000% 	           0
low-Q discards:  	  0.0137% 	        5 	  0.0137% 	         630

perfect best site:	  0.0000% 	        0 	  0.0000% 	           0
semiperfect site:	  0.0000% 	        0 	  0.0000% 	           0
rescued:         	  0.0220% 	        8

Match Rate:      	      NA 	       NA 	 84.0716% 	        4555
Error Rate:      	100.0000% 	       43 	 15.8915% 	         861
Sub Rate:        	100.0000% 	       43 	 15.2270% 	         825
Del Rate:        	  0.0000% 	        0 	  0.0000% 	           0
Ins Rate:        	 16.2791% 	        7 	  0.6645% 	          36
N Rate:          	  4.6512% 	        2 	  0.0369% 	           2


Read 2 data:      	pct reads	num reads 	pct bases	   num bases

mapped:          	  0.1428% 	       52 	  0.1428% 	        6552
unambiguous:     	  0.1428% 	       52 	  0.1428% 	        6552
ambiguous:       	  0.0000% 	        0 	  0.0000% 	           0
low-Q discards:  	  1.6751% 	      610 	  1.6751% 	       76860

perfect best site:	  0.0000% 	        0 	  0.0000% 	           0
semiperfect site:	  0.0000% 	        0 	  0.0000% 	           0
rescued:         	  0.0275% 	       10

Match Rate:      	      NA 	       NA 	 84.1270% 	        5512
Error Rate:      	100.0000% 	       52 	 15.8730% 	        1040
Sub Rate:        	100.0000% 	       52 	 15.8730% 	        1040
Del Rate:        	  0.0000% 	        0 	  0.0000% 	           0
Ins Rate:        	  0.0000% 	        0 	  0.0000% 	           0
N Rate:          	  0.0000% 	        0 	  0.0000% 	           0

[GenoMax]

That does not look promising. Few reads are mapped.

You are going to have to spend sometime playing with BBMap options to see if you can improve the alignments. Checking some of the individual reads would be needed to see if you have chimeric reads and/or just random hits based on the initial selection of k=25. Have you tried using higher k values to see if you can narrow down the read set?

[ErikFas]

Actually for this data (i.e. RNA-seq) the sequence shouldn't be there, so aligning few reads is actually good for me! I will run through the workflow again with another k-mer, maybe 35 or 50?

I looked at the BAM file in IGV, and it looks like the attached image. If I'm interpreting this correctly, there are a bunch of reads that map to the sequence, but they all have a bunch of mutations, which would then correspond to the allowed mismatched in the BBDuk step? Should I then rerun with fewer (or no) allowed mismatches, or am I misinterpreting the data?

I would also like to do this on my whole genome sequencing data, would I just do the exact same procedure for that (starting at the fastq-files from DNA sequencing)?