Skip to content
New issue

Have a question about this project? Sign up for a free GitHub account to open an issue and contact its maintainers and the community.

By clicking “Sign up for GitHub”, you agree to our terms of service and privacy statement. We’ll occasionally send you account related emails.

Already on GitHub? Sign in to your account

Tracer assemble gives two chains with identical CDR3 sequence as different #118

Open
diitaz93 opened this issue Apr 5, 2022 · 0 comments

Comments

@diitaz93
Copy link

diitaz93 commented Apr 5, 2022

Hi!
Thanks for developing Tracer!
I am using it with a dataset of ~4k cells and there is something strange with the output of assemble with one specific cell. The file filtered_TCRs.txt outputs two Beta chains but both have the same ID (see below). When you look closely you can note the following:

  • Both chains share the CDR3 sequence, the D and the J segment.
  • The V segment only differs in the allele of the usage (TRBV7-8*01 vs. TRBV7-8*03)
  • The TPM of the first cell is unusually small (3.29) while the second one has values similar to other detected chains in my dataset (1472)
  • In both chains the TRBV7-8*01 has 100 % identity, while TRBV7-8*03 has 99.286 and 99.303, respectively. However *03 allele was chosen in the first chain.

I don't know if this is unexpected behaviour. Should I assume both chains are the same? or is there a significant difference between the two sequences?
The output of unfiltered_TCR.txt is exactly the same as filtered_TCR.txt.

#TCR_B#
##TRINITY_DN0_c0_g1_i2##
V segment:      TRBV7-8*03
D segment:      TRBD1*01
J segment:      TRBJ2-2*01
ID:     TRBV7-8_AGACGCTCAGGGGTGGTCACCG_TRBJ2-2
TPM:    3.29375
Productive:     True
Stop codon:     False
In frame:       True
CDR3aa: ASRRSGVVTGELF
CDR3nt: GCCAGCAGACGCTCAGGGGTGGTCACCGGGGAGCTGTTT

Segment query_id        subject_id      % identity      alignment length        mismatches      gap opens       gaps
    q start q end   s start s end   e value bit score
V       reversed|TRINITY_DN0_c0_g1_i2   TRBV7-8*03      99.286  140     1       0       0       78      217     148
     287     2.40e-58        216
V       reversed|TRINITY_DN0_c0_g1_i2   TRBV7-8*01      100.000 137     0       0       0       78      214     148
     284     7.08e-58        215
V       reversed|TRINITY_DN0_c0_g1_i2   TRBV7-8*02      99.270  137     1       0       0       78      214     148
     284     6.14e-57        212
V       reversed|TRINITY_DN0_c0_g1_i2   TRBV7-4*01      89.362  141     15      0       0       74      214     144
     284     1.11e-45        174
V       reversed|TRINITY_DN0_c0_g1_i2   TRBV7-2*01      93.496  123     8       0       0       92      214     162
     284     8.34e-44        168
D       reversed|TRINITY_DN0_c0_g1_i2   TRBD1*01        100.000 6       0       0       0       220     225     5
       10      4.0     12.4
J       reversed|TRINITY_DN0_c0_g1_i2   TRBJ2-2*01      100.000 47      0       0       0       230     276     5
       51      1.93e-22        93.7


##TRINITY_DN0_c0_g1_i1##
V segment:      TRBV7-8*01
D segment:      TRBD1*01
J segment:      TRBJ2-2*01
ID:     TRBV7-8_AGCAGACGCTCAGGGGTGGTCACCG_TRBJ2-2
TPM:    1472.31
Productive:     True
Stop codon:     False
In frame:       True
CDR3aa: ASRRSGVVTGELF
CDR3nt: GCCAGCAGACGCTCAGGGGTGGTCACCGGGGAGCTGTTT

Segment query_id        subject_id      % identity      alignment length        mismatches      gap opens       gaps
    q start q end   s start s end   e value bit score
V       reversed|TRINITY_DN0_c0_g1_i1   TRBV7-8*01      100.000 284     0       0       0       16      299     1
       284     8.99e-127       444
V       reversed|TRINITY_DN0_c0_g1_i1   TRBV7-8*03      99.303  287     2       0       0       16      302     1
       287     2.65e-126       442
V       reversed|TRINITY_DN0_c0_g1_i1   TRBV7-8*02      99.648  284     1       0       0       16      299     1
       284     7.80e-126       441
V       reversed|TRINITY_DN0_c0_g1_i1   TRBV7-6*01      90.845  284     26      0       0       16      299     1
       284     2.21e-102       363
V       reversed|TRINITY_DN0_c0_g1_i1   TRBV7-4*01      90.493  284     27      0       0       16      299     1
       284     1.91e-101       360
D       reversed|TRINITY_DN0_c0_g1_i1   TRBD1*01        100.000 6       0       0       0       305     310     5
       10      4.5     12.4
J       reversed|TRINITY_DN0_c0_g1_i1   TRBJ2-2*01      100.000 47      0       0       0       315     361     5
       51      2.17e-22        93.7

Sign up for free to join this conversation on GitHub. Already have an account? Sign in to comment
Labels
None yet
Projects
None yet
Development

No branches or pull requests

1 participant