Hi,
We recently ran TRUST4 on some 10x Genomics TCR data using the command
trust4 -f hg38_bcrtcr.fa --ref human_IMGT+C.fa -1 input/YWI006-VDJ_S1_L001_R1_001.fastq.gz -2 input/VDJ/YWI006-VDJ_S1_L001_R2_001.fastq.gz --barcode input/VDJ/YWI006-VDJ_S1_L001_R1_001.fastq.gz --readFormat bc:0:15,r1:16:-1 -o /nethome/daniel.fonseca/Documents/trust4/output/
We are currently benchmarking TRUST4 against MiXCR and CellRangerVDJ so are comparing the results across all 3 softwares and have a couple of questions regarding the TRUST4 output.
TRUST4 identifies a significantly greater number of unique clones compared to MiXCR and CellRangerVDJ .
CellRangerVDJ unique clones: 17
MiXCR v4 unique clones: 79
TRUST4 unique clones: 412
One reason is that some of clones may have point mutations after PCR amplification, so because of this we calculated the levenshtein distance between the concatenated alpha and beta cdr3 nucleotide sequences. We collapsed clones with a distance of either 1 or 2 for all software’s. We found that TRUST4 still had a much greater number of unique clones.
CellRangerVDJ unique clones: 16
MiXCR v4 unique clones: 55
TRUST4 unique clones:167
So first, we were wondering what sort of pre filtering is done within the TRUST4 algorithm on the sequences before getting the final output, and if not, what quality control measures should we be taking to ensure we are keeping real hits.
Second, many of the clones in TRUST4 have only 1 read, is it sensible to keep these? Or should we implementing some sort of read cut off?
Additionally, there is a read and a umi column in the final output, and we were wondering what exactly is the different because the values in both of these columns is the same.
Finally, when looking at overlap of these clones between the different software’s we find that many clones (especially between TRUST4 and MiXCR) do not overlap (see plot below). Do you have any thoughts as to why this could be?
Hi, We recently ran TRUST4 on some 10x Genomics TCR data using the command
trust4 -f hg38_bcrtcr.fa --ref human_IMGT+C.fa -1 input/YWI006-VDJ_S1_L001_R1_001.fastq.gz -2 input/VDJ/YWI006-VDJ_S1_L001_R2_001.fastq.gz --barcode input/VDJ/YWI006-VDJ_S1_L001_R1_001.fastq.gz --readFormat bc:0:15,r1:16:-1 -o /nethome/daniel.fonseca/Documents/trust4/output/We are currently benchmarking TRUST4 against MiXCR and CellRangerVDJ so are comparing the results across all 3 softwares and have a couple of questions regarding the TRUST4 output. TRUST4 identifies a significantly greater number of unique clones compared to MiXCR and CellRangerVDJ .
One reason is that some of clones may have point mutations after PCR amplification, so because of this we calculated the levenshtein distance between the concatenated alpha and beta cdr3 nucleotide sequences. We collapsed clones with a distance of either 1 or 2 for all software’s. We found that TRUST4 still had a much greater number of unique clones.
So first, we were wondering what sort of pre filtering is done within the TRUST4 algorithm on the sequences before getting the final output, and if not, what quality control measures should we be taking to ensure we are keeping real hits.
Second, many of the clones in TRUST4 have only 1 read, is it sensible to keep these? Or should we implementing some sort of read cut off?
Additionally, there is a read and a umi column in the final output, and we were wondering what exactly is the different because the values in both of these columns is the same.
Finally, when looking at overlap of these clones between the different software’s we find that many clones (especially between TRUST4 and MiXCR) do not overlap (see plot below). Do you have any thoughts as to why this could be?
Thanks in advance for your response :)