Reverse primers

Manufactured by Adaptive Biotechnologies

Reverse primers are short nucleic acid sequences used in polymerase chain reaction (PCR) methods. They bind to the complementary strand of a DNA template, allowing the amplification of specific target sequences.

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Lab products found in correlation

Survey immunoseq assay, by Adaptive Biotechnologies (2 mentions) Forward primers, by Adaptive Biotechnologies (2 mentions) Ffpe dna kit, by Qiagen (1 mentions)

2 protocols using reverse primers

Evaluation of TCR Clones in Tumor Tissue

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TCR clones were evaluated in tumor tissue by NGS. DNA from tumor samples was isolated by using the Qiagen DNA FFPE kit. TCR-β CDR3 regions were amplified using the survey ImmunoSeq assay in a multiplex PCR method using 45 forward primers specific to TCR Vβ gene segments and 13 reverse primers specific to TCR Jβ gene segments (Adaptive Biotechnologies)^{62 (link)}. Productive TCR sequences were further analyzed. For each sample, a clonality metric was estimated to quantitate the extent of mono- or oligo-clonal expansion by measuring the shape of the clone frequency distribution. Clonality values range from 0 to 1, where values approaching 1 indicate a nearly monoclonal population (Supplementary Table 13).

Anagnostou V., Niknafs N., Marrone K., Bruhm D.C., White J.R., Naidoo J., Hummelink K., Monkhorst K., Lalezari F., Lanis M., Rosner S., Reuss J.E., Smith K.N., Adleff V., Rodgers K., Belcaid Z., Rhymee L., Levy B., Feliciano J., Hann C.L., Ettinger D.S., Georgiades C., Verde F., Illei P., Li Q.K., Baras A.S., Gabrielson E., Brock M.V., Karchin R., Pardoll D.M., Baylin S.B., Brahmer J.R., Scharpf R.B., Forde P.M, & Velculescu V.E. (2020). Multimodal genomic features predict outcome of immune checkpoint blockade in non-small-cell lung cancer. Nature cancer, 1(1), 99-111.

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Quantitative TCR Profiling of Tumor Samples

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TCR clones were evaluated in tumor tissue by next generation sequencing, with an input material of 500ng of genomic DNA for each sample analyzed. TCR-β CDR3 regions were amplified using the survey ImmunoSeq assay in a multiplex PCR method using 45 forward primers specific to TCR Vβ gene segments and 13 reverse primers specific to TCR Jβ gene segments (Adaptive Biotechnologies)⁶² (link). Productive TCR sequences were further analyzed (Table S1H). For each sample, a clonality metric was estimated in order to quantitate the extent of mono- or oligo-clonal expansion by measuring the shape of the clone frequency distribution. Clonality values range from 0 to 1, where values approaching 1 indicate a nearly monoclonal population (Table S1H). For differential abundance analysis between baseline and on-therapy tumors, we selected the most expanded and most regressed TCR clonotypes, corresponding to fold changes in productive frequency of TCR clones with an FDR < 0.001. These TCR clones are referred to as expanded and regressed.

Anagnostou V., Bruhm D.C., Niknafs N., White J.R., Shao X.M., Sidhom J.W., Stein J., Tsai H.L., Wang H., Belcaid Z., Murray J., Balan A., Ferreira L., Ross-Macdonald P., Wind-Rotolo M., Baras A.S., Taube J., Karchin R., Scharpf R.B., Grasso C., Ribas A., Pardoll D.M., Topalian S.L, & Velculescu V.E. (2020). Integrative Tumor and Immune Cell Multi-omic Analyses Predict Response to Immune Checkpoint Blockade in Melanoma. Cell Reports Medicine, 1(8), 100139.

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