12% gamma chain V,J, CDR3aa coverage

[tiannagrant]

Hello,

I used TRUST4 to align VDJ region of gamma delta T cells. I used the cellranger output sample_alignments.bam file to align unassigned sequences

#the code Tianna ran 
run-trust4 -b /path_to_bam/Control/count/sample_alignments.bam -f 
/path_to_reference/TRUST4/mouse/GRCm38_bcrtcr.fa --ref path_to_IMGT_reference/mouse/mouse_IMGT+C.fa --barcode CB

Cellranger GEX:barcode I have an 15,000 cells but TRUST4 only found TCRs for 12% or cells ~1200 cells. Also, I expected a specific cluster to be enriched for a specific Variable gene on the gamma chain but I'm not observing that. Have you seen this in your dataset? what are some troubleshoot workflows you recommend?

[mourisl]

Is your single-cell data 5'end or 3'end?

[tiannagrant]

Hi Li,

5' end.

Best, Tianna

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From: Li Song @.> Sent: Monday, March 20, 2023 2:57 PM To: liulab-dfci/TRUST4 @.> Cc: Grant, Tianna @.>; Author @.> Subject: Re: [liulab-dfci/TRUST4] 12% gamma chain V,J, CDR3aa coverage (Issue #189)

Is your single-cell data 5'end or 3'end? — Reply to this email directly, view it on GitHub, or unsubscribe. You are receiving this because you authored the thread. Message ID: liulab-dfci/TRUST4/issues/189/1476988631@ github. com ZjQcmQRYFpfptBannerStart This Message Is From an External Sender This message came from outside your organization.

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Is your single-cell data 5'end or 3'end?

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[mourisl]

I guess you may have a relatively low gene coverage within each cell to reach the 15K number. What is the average number of reads in the QC-passed cell? How many T cells and B cells are in the sample?

[tiannagrant]

I QC'ed cellranger output of GEX object using median absolute deviation. My average total counts for sample A: 5327 sample B: 11706 I would say using 5' 10X kit should produce great VDJ coverage compared to 3'.

According TRUST4: prior to defining gdTCR clonotypes gdT : 5602 B:1738 abT :1418

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From: Li Song @.> Sent: Monday, March 20, 2023 3:59 PM To: liulab-dfci/TRUST4 @.> Cc: Grant, Tianna @.>; Author @.> Subject: Re: [liulab-dfci/TRUST4] 12% gamma chain V,J, CDR3aa coverage (Issue #189)

I guess you may have a relatively low gene coverage within each cell to reach the 15K number. What is the average number of reads in the QC-passed cell? How many T cells and B cells are in the sample? — Reply to this email directly, view ZjQcmQRYFpfptBannerStart This Message Is From an External Sender This message came from outside your organization.

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I guess you may have a relatively low gene coverage within each cell to reach the 15K number. What is the average number of reads in the QC-passed cell? How many T cells and B cells are in the sample?

— Reply to this email directly, view it on GitHubhttps://urldefense.com/v3/__https://github.com/liulab-dfci/TRUST4/issues/189*issuecomment-1477058386__;Iw!!LQC6Cpwp!rcT5EX8S_8562kEbJrD7CGw5abBrK5jhDW_BtKgrl2WpecKMjoHNZ3OesatWtrXTAKTRfK-XLqhZffguJ7G8P9jHn5M$, or unsubscribehttps://urldefense.com/v3/__https://github.com/notifications/unsubscribe-auth/ANC6TFNUA77FT5JNVYPTPVTW5DONFANCNFSM6AAAAAAWBUYD5A__;!!LQC6Cpwp!rcT5EX8S_8562kEbJrD7CGw5abBrK5jhDW_BtKgrl2WpecKMjoHNZ3OesatWtrXTAKTRfK-XLqhZffguJ7G89GqmL7g$. You are receiving this because you authored the thread.Message ID: @.***>

[mourisl]

I think the typical read count/cell in 10X is around 20K~30K, so the coverage of your data set is indeed low, especially in sample A. If we have no read from the VDJ region, it is impossible to find the sequences.

For the second part, do you mean out of the 5602 gdT cells called from TRUST4, none of them overlap with your annotated gdT inferred from GEX?

[HACKYANG123]

Hi Tianna I also want to detect T cells with gamma-delta chains in my V(D)J data. I had previously used custom primers (Gherardin, N. A., Waldeck, K., Caneborg, A. et al. γδ T cells in Merkel cell carcinomas have a proinflammatory profile prognostic of patient survival. Cancer Immunology Research 9, 612-623) to amplify TRG/D chains by using the Chromium Next GEM Single Cell 5' Assay kit. I used Cell Ranger (v7.0) to process data of TCR libraries enriched for TRG/D chains. But most of the contig were filtered out because is_cell false and high_confidence false. However, I can see many barcode that are actually true cells in gene expression data are filtered out in Filtered_contig_annotations.csv (file://xn--1-471bx41aba436x/#contig). Furthermore, since TRGV9 chain are always paired with TRDV2 chain, it seems that these contig are actually have high confidence. I don’t know why so many contig were filtered out because is_cell false and high_confidence false? ,Have you ever met a similar situation? what algorithm did you finnally use to annotate gdTCR? TRUST4/ Cell Ranger/Dandelion? Best, Yan Yang

[HACKYANG123]

Dear Dr.mourisl,

I want to detect γδ TCR in my 10x 5’ scRNA data. I had previously used custom primers to amplify TRG/D chains by using the Chromium Next GEM Single Cell 5' Assay kit. I used TRUST4 to assemble γδ TCRS. Can I assume that all contigs in the output are high confident? But I think some contigs are false positive, some contigs are not full length and the number of count in many contigs is only 1(I have attached output file). Do I need to filter out some contig with low confidence by myself?

Best,

Yan