kallisto bus parsing barcode length to be 0

[caleblareau]

Hi kallisto team,

I'm trying to quantify gRNAs for a scRNA/Perturb-seq experiment. The barcode/umi/gRNA are on a single-end read.

I've created an index using kite and then attempted to use kallisto|bustools but am getting a weird error where it appears that the parsed barcode length is 0.

Running

kallisto bus -i gRNA_kallisto_index test.fastq.gz -o bus_out -x 0,0,16:0,16,26:0,38,60

seems fine (stdout below):

[index] k-mer length: 21
[index] number of targets: 9,984
[index] number of k-mers: 9,951
[index] number of equivalence classes: 9,997
[quant] will process sample 1: test.fastq.gz
[quant] finding pseudoalignments for the reads ... done
[quant] processed 25,000 reads, 2,630 reads pseudoaligned

However, when I run the bustools correct command:

bustools correct -w data/737K-august-2016.txt -o corrected_bus_out bus_out

I get the following error:

Found 737280 barcodes in the whitelist
Error: barcode length and whitelist length differ, barcodes = 0, whitelist = 16
       check that your whitelist matches the technology used

My understanding is that the -x parameter should have correctly specified a 16bp barcode. Attempting to skip the bustools correct step leads to a similar error downstream (in bustools sort) as well.

My fastq file for reference:

zcat < test.fastq.gz | head
@NS500466:533:H5HNJBGXF:1:11101:20629:1041 1:N:0:TGGTAACG
GCTCCNAAGTAGATGTCCCAATTCAATTTCTTATATNGGGTCCGTTATAACTTGAAAAANTGGCACCGGTCGGTAG
+
AAAAA#EEEEEEEEEEEEEEEEEAEEEEEEEEEEEE#EEEEEAEEEEEEEEEEEEEEEE#EEA<AEEEEEEEA6AA
@NS500466:533:H5HNJBGXF:1:11101:1592:1041 1:N:0:TGGTAACG
CGCTGNACAGTAACGGACGCCACGGGTTTCTTATATNGGGTTATCAACTTGAAAAAGTGNCACCGAGTCGGTAGAT
+
AAAAA#EEEEEEEE/EEEEEAEEEEAEAEEEEAEEE#EAEEEEEEEEEAAAA//AEEEE#EEEEEE/EE/EE<E</
@NS500466:533:H5HNJBGXF:1:11101:15220:1042 1:N:0:TGGTAACG
AACTCNCTCACTCTTACTCGGTCGTCTTTCTTATATGGGTCAACTTGAAAAAGTGGCACNGAGTCGGTAGATCGGA

Any clues as to what's happening? I've also tried splitting things into multiple files (and updating -x), but I get a similar "barcodes = 0" error.

I'm using kallisto 0.46.1 and bustools 0.40.0.

Any help would be greatly appreciated. Thank you!