Confronting false discoveries in single-cell differential expression methods
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Application to spinal cord injury
Experiments were conducted on adult male or female C57BL/6 mice (15-35 g body weight, 12-30 weeks of age). Vglut2:Cre (Jackson Laboratory 016963) transgenic mice were used and maintained on a mixed genetic background (129/C57BL/6). Housing, surgery, behavioral experiments and euthanasia were performed in compliance with the Swiss Veterinary Law guidelines. Animal care, including manual bladder voiding, was performed twice daily for the first 3 weeks after injury and once daily for the remaining post-injury period. All procedures and surgeries were approved by the Veterinary Office of the Canton of Geneva (Switzerland; GE/57/20 A).
Surgical procedures and post-surgical care
Surgical procedures were performed as previously described, -. Briefly, a laminectomy was made at the mid-thoracic level (T9 vertebra). We performed a contusion injury using a force-controlled spinal cord impactor (IH-0400 Impactor, Precision Systems and Instrumentation LLC, USA ), as previously described,. The applied force was set to 90 kdyn. Analgesia (buprenorphine, Essex Chemie AG, Switzerland, 0.01-0.05 mg per kg, s.c.) was provided for three days after surgery.
Mixed models
Having established that the performance of DE methods is contingent on their ability to account for biological replicates, we asked why mixed models failed to match the performance of pseudobulk methods. In addition to the linear mixed model described above, we implemented generalized linear mixed models (GLMMs) based on the negative binomial or Poisson distributions, adapting implementations provided in the 'muscat' R package. For each of these models, we evaluated the impact of incorporating the library size factors as an offset term, and compared the Wald test of model coefficients to a likelihood ratio test against a reduced model, yielding a total of four GLMMs from each distribution. The enormous computational requirements of the GLMMs prevented us from evaluating these models in the full ground truth datasets; instead, we analyzed a series of downsampled datasets,...
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Experiments were conducted on adult male or female C57BL/6 mice (15-35 g body weight, 12-30 weeks of age). Vglut2:Cre (Jackson Laboratory 016963) transgenic mice were used and maintained on a mixed genetic background (129/C57BL/6). Housing, surgery, behavioral experiments and euthanasia were performed in compliance with the Swiss Veterinary Law guidelines. Animal care, including manual bladder voiding, was performed twice daily for the first 3 weeks after injury and once daily for the remaining post-injury period. All procedures and surgeries were approved by the Veterinary Office of the Canton of Geneva (Switzerland; GE/57/20 A).
Surgical procedures were performed as previously described, -. Briefly, a laminectomy was made at the mid-thoracic level (T9 vertebra). We performed a contusion injury using a force-controlled spinal cord impactor (IH-0400 Impactor, Precision Systems and Instrumentation LLC, USA ), as previously described,. The applied force was set to 90 kdyn. Analgesia (buprenorphine, Essex Chemie AG, Switzerland, 0.01-0.05 mg per kg, s.c.) was provided for three days after surgery.
Having established that the performance of DE methods is contingent on their ability to account for biological replicates, we asked why mixed models failed to match the performance of pseudobulk methods. In addition to the linear mixed model described above, we implemented generalized linear mixed models (GLMMs) based on the negative binomial or Poisson distributions, adapting implementations provided in the 'muscat' R package. For each of these models, we evaluated the impact of incorporating the library size factors as an offset term, and compared the Wald test of model coefficients to a likelihood ratio test against a reduced model, yielding a total of four GLMMs from each distribution. The enormous computational requirements of the GLMMs prevented us from evaluating these models in the full ground truth datasets; instead, we analyzed a series of downsampled datasets, each containing between 25 and 1,000 cells. To quantify the computational resources required by each DE method, we monitored peak memory usage using the 'peakRAM' R package, and the base R function 'system.time' to record wall time.
Machine-readable layer
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