Hi, I'm using kallisto and bustools for single cell RNA-seq, in a similar way to what is done in the Pall Melsted et. al. paper here: https://www.biorxiv.org/content/10.1101/673285v1 I'm trying to get an idea of what information is in the reads that aren't aligned to anything, and get counts of the sequences that don't align to anything but appear frequently in the fastq. I've used CITE-seq-Count for a similar purpose before, and it creates an unmapped.csv file in its output, which is exactly what I'm looking for. However, I've been given a requirement to not use CITE-seq-Count for this project. Is there a way to get the reads that don't map to anything from the output of the kallisto bus command? Or alternatively, if I could get the ID's of the reads that do match, I could subset them out of the original fastq and use fastqc to get a list of overrrepresented sequences and their abundances in the remaining. Any advice would be greatly appreciated!

Running kallisto bus with the -n option will record the read numbers of the reads that have been successfully mapped in the output BUS file. You can inspect the BUS file in standard output using bustools text -pf /path/to/output/file.bus -- the last column will contain the read numbers (zero-indexed) of every read that has been successfully mapped. From there, you can figure out what the unmapped read numbers are and then go into your FASTQ file to pick out those sequences.