Comparison of downstream data quality of two differential extraction techniques through the examination of peak heights and peak height ratios

Analysis of sexual assault evidence in a forensic laboratory setting requires accurate analysis of deoxyribonucleic acid (DNA) utilizing forensic DNA typing. This process can be complicated since most sexual assault samples are comprised of DNA from a female contributor as well as one or more male contributors. Generally female epithelial cells (e-cells) are in excess, making it more difficult to determine the short tandem repeat (STR) profile of the male contributor from the significantly fewer sperm cells present in the mixture. This complexity requires that the two contributing sources of DNA be separated in order to obtain a probative single source profile. Separation of the two fractions is accomplished by differential extraction. The most common protocol for a differential extraction involves preferentially lysing epithelial cells while leaving the sperm cells intact, separating the released female DNA from the sperm cells and finally lysing the sperm cells to obtain the sperm fraction. Research has shown that a Trypsin-ZyGEM extraction method produces higher yields of DNA compared to the standard differential extraction method. A previous study examined whether substrate type had an effect on the efficiency of the Trypsin-ZyGEM extraction. It was found that the Trypsin-ZyGEM protocol worked well at extracting DNA from aqueous semen, dried semen in a microfuge tube and semen dried on white cotton. However, results were inconclusive on the method’s ability to extract DNA from semen dried on denim fabric due to inhibition of the quantitative polymerase chain reaction (qPCR) used to quantify the samples. In this study, a subset of the denim samples was amplified and processed through capillary electrophoresis to verify that the extraction protocol had successfully recovered DNA from this substrate type and that the DNA could be amplified to create a STR profile. Full profiles were obtained for all three of the denim samples that were amplified to a target mass of 1 nanogram (ng). One full profile and two partial profiles were obtained for the same samples at a target mass of 0.25ng. Allelic dropout for the partial profiles varied with one profile having 3 alleles dropout while the other had 11 dropout. Peak heights and peak height ratios were more variable compared to the other substrate types but were still within an acceptable range for probative profiles. The results are promising and suggest that the Trypsin-ZyGEM protocol could possibly replace the current differential extraction technique. Further research is required to understand if this extraction protocol would be efficient on these substrate types with mixture samples of semen and e-cells. This research also builds upon the exploration of the effectiveness of the Trypsin-ZyGEM extraction on dried and aqueous semen samples compared to a modified Qiagen differential extraction. Samples that had been previously quantified were amplified and separated by size using capillary electrophoresis. Peak heights and peak height ratios (PHR) from the STR profiles were examined to assess profile quality of samples extracted using the Trypsin-ZyGEM protocol and the Qiagen protocol. Peak heights and peak height ratios (PHR) were found to be consistent between the two methods.2017-11-03T00:00:00

The immune cell landscape in kidneys of patients with lupus nephritis.

Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies

Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue

Abstract Background Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. Methods Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. Results Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 μg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. Conclusions We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers

The immune cell landscape in kidneys of patients with lupus nephritis.

Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies