The data was aligned successfully, but assemble outputs 'Final clonotype count: 0'

[linqy-immune]

Hello, I am a big fan of mixcr and I have processed some data by mixcr. Recently, however, when I try to processed a set of data using "analyze" parameter, it happened that the align ouput seemed as usual like this "Total sequencing reads: 10136933
Successfully aligned reads: 7596017 (74.93%)
Coverage (percent of successfully aligned):
CDR3: 7572819 (99.69%)
FR3_TO_FR4: 460 (0.01%)
CDR2_TO_FR4: 460 (0.01%)
FR2_TO_FR4: 26 (0%)
CDR1_TO_FR4: 0 (0%)
VDJRegion: 0 (0%)
Alignment failed: no hits (not TCR/IG?): 1452482 (14.33%)
Alignment failed after alignment-aided overlap: 10 (0%)
Alignment failed: absence of V hits: 402200 (3.97%)
Alignment failed: absence of J hits: 77413 (0.76%)
Alignment failed: no target with both V and J alignments: 608811 (6.01%)
Overlapped: 1273090 (12.56%)
Overlapped and aligned: 124314 (1.23%)
Overlapped and not aligned: 1148776 (11.33%)
Alignment-aided overlaps, percent of overlapped and aligned: 18749 (15.08%)
No CDR3 parts alignments, percent of successfully aligned: 1598 (0.02%)
Partial aligned reads, percent of successfully aligned: 21600 (0.28%)
V gene chimeras: 117725 (1.16%)
J gene chimeras: 4 (0%)
Paired-end alignment conflicts eliminated: 2 (0%)
Realigned with forced non-floating bound: 8882592 (87.63%)
Realigned with forced non-floating right bound in left read: 109831 (1.08%)
Realigned with forced non-floating left bound in right read: 109831 (1.08%)
TRA chains: 63 (0%)
TRA non-functional: 0 (0%)
TRB chains: 7595954 (100%)
TRB non-functional: 229735 (3.02%)
Trimming report:
R1 reads trimmed left: 28345 (0.28%)
R1 reads trimmed right: 699859 (6.9%)
Average R1 nucleotides trimmed left: 0.019284728428213938
Average R1 nucleotides trimmed right: 1.338412713194415
R2 reads trimmed left: 123093 (1.21%)
R2 reads trimmed right: 1846162 (18.21%)
Average R2 nucleotides trimmed left: 0.053884838737712874
Average R2 nucleotides trimmed right: 4.9276925279076025",
but the assemble output was like this "Final clonotype count: 0
Reads used in clonotypes, percent of total: 0 (0%)
Average number of reads per clonotype: NaN
Reads dropped due to the lack of a clone sequence, percent of total: 7596017 (74.93%)
Reads dropped due to a too short clonal sequence, percent of total: 0 (0%)
Reads dropped due to low quality, percent of total: 0 (0%)
Reads dropped due to failed mapping, percent of total: 0 (0%)
Reads dropped with low quality clones, percent of total: 0 (0%)
Aligned reads processed: 0
Reads used in clonotypes before clustering, percent of total: 0 (0%)
Number of reads used as a core, percent of used: 0 (NaN%)
Mapped low quality reads, percent of used: 0 (NaN%)
Reads clustered in PCR error correction, percent of used: 0 (NaN%)
Reads pre-clustered due to the similar VJC-lists, percent of used: 0 (NaN%)
Clonotypes dropped as low quality: 0
Clonotypes eliminated by PCR error correction: 0
Clonotypes pre-clustered due to the similar VJC-lists: 0
Clones dropped in post filtering: 0 (NaN%)
Reads dropped in post filtering: 0.0 (0%)
Reads filtered by tag prefix: 0 (0%)", as a result, the export.clones was empty but only showed the titles.
The command I ran was "mixcr analyze illumina-human-rna-trb-ampliseq-plus SRR5812610_1.fastq SRR5812610_2.fastq result1" and the software version was mixcr4.7.0.
I was stuck on this question and made no progress for my bioinformatics, so I tried to ask you for help. Thank you very much!!!
Yours sincerely, Linqy

[mizraelson]

Hi Linqy
It looks like you have data generated using the short-read kit, but you used the long-read preset, which expect other regions to be covered in addition to CDR3. For your data, please use: illumina-human-rna-trb-ampliseq-sr.