Bias, robustness and scalability in differential expression analysis of single-cell RNA-seq data, bioRxiv, 2017-05-29

AbstractBackgroundAs single-cell RNA-seq (scRNA-seq) is becoming increasingly common, the amount of publicly available data grows rapidly, generating a useful resource for computational method development and extension of published results. Although processed data matrices are typically made available in public repositories, the procedure to obtain these varies widely between data sets, which may complicate reuse and cross-data set comparison. Moreover, while many statistical methods for performing differential expression analysis of scRNA-seq data are becoming available, their relative merits and the performance compared to methods developed for bulk RNA-seq data are not sufficiently well understood.ResultsWe present conquer, a collection of consistently processed, analysis-ready public single-cell RNA-seq data sets. Each data set has count and transcripts per million (TPM) estimates for genes and transcripts, as well as quality control and exploratory analysis reports. We use a subset of the data sets available in conquer to perform an extensive evaluation of the performance and characteristics of statistical methods for differential gene expression analysis, evaluating a total of 30 statistical approaches on both experimental and simulated scRNA-seq data.ConclusionsConsiderable differences are found between the methods in terms of the number and characteristics of the genes that are called differentially expressed. Pre-filtering of lowly expressed genes can have important effects on the results, particularly for some of the methods originally developed for analysis of bulk RNA-seq data. Generally, however, methods developed for bulk RNA-seq analysis do not perform notably worse than those developed specifically for scRNA-seq.

biorxiv bioinformatics 100-200-users 2017

Achieving better than 3 Å resolution by single particle cryo-EM at 200 keV, bioRxiv, 2017-05-26

AbstractTechnical and methodological advances in single-particle cryo-electron microscopy (cryo-EM) have expanded the technique into a resolution regime that was previously only attainable by X-ray crystallography. Although single-particle cryo-EM has proven to be a useful technique for determining the structures of biomedically relevant molecules at near-atomic resolution, nearly 98% of the structures resolved to better than 4 Å resolution have been determined using 300 keV transmission electron microscopes (TEMs). We demonstrate that it is possible to obtain cryo-EM reconstructions of macromolecular complexes at a range of sizes to better than 3 Å resolution using a 200 keV TEM. These structures are of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules, features previously thought only to be resolvable using TEMs operating at 300 keV.

biorxiv biophysics 100-200-users 2017

Single-cell RNA-seq of rheumatoid arthritis synovial tissue using low cost microfluidic instrumentation, bioRxiv, 2017-05-23

AbstractDroplet-based single cell RNA-seq has emerged as a powerful technique for massively parallel cellular profiling. While these approaches offer the exciting promise to deconvolute cellular heterogeneity in diseased tissues, the lack of cost-effective, reliable, and user-friendly instrumentation has hindered widespread adoption of droplet microfluidic techniques. To address this, we have developed a microfluidic control instrument that can be easily assembled from 3D printed parts and commercially available components costing approximately $540. We adapted this instrument for massively parallel scRNA-seq and deployed it in a clinical environment to perform single cell transcriptome profiling of disaggregated synovial tissue from a rheumatoid arthritis patient. We sequenced 8,716 single cells from a synovectomy, revealing 16 transcriptomically distinct clusters. These encompass a comprehensive and unbiased characterization of the autoimmune infiltrate, including inflammatory T and NK subsets that contribute to disease biology. Additionally, we identified fibroblast subpopulations that are demarcated via THY1 (CD90) and CD55 expression. Further experiments confirm that these represent synovial fibroblasts residing within the synovial intimal lining and subintimal lining, respectively, each under the influence of differing microenvironments. We envision that this instrument will have broad utility in basic and clinical settings, enabling low-cost and routine application of microfluidic techniques, and in particular single-cell transcriptome profiling.

biorxiv genomics 100-200-users 2017

Manuscript 101 a data-driven writing exercise for beginning scientists, bioRxiv, 2017-05-19

AbstractLearning to write a scientific manuscript is one of the most important and rewarding scientific training experiences, yet most young scientists only embark on this experience relatively late in graduate school, after gathering sufficient data in the lab. Yet, familiarity with the process of writing a scientific manuscript and receiving peer reviews, often leads to a more focused and driven experimental approach. To jump-start this training, we developed a protocol for teaching manuscript writing and reviewing in the classroom, appropriate for new graduate or upper-level undergraduate students of developmental biology. First, students are provided one of four cartoon data sets, which are focused on genetic models of animal development. Students are instructed to use their creativity to convert evidence into argument, and then to integrate their interpretations into a manuscript, including an illustrated, mechanistic model figure. After student manuscripts are submitted, manuscripts are redacted and distributed to classmates for peer review. Here, we present our cartoon datasets, homework instructions, and grading rubrics as a new resource for the scientific community. We also describe methods for developing new datasets so that instructors can adapt this activity to other disciplines. Our data-driven manuscript writing exercise, as well as the formative and summative assessments resulting from the peer review, enables students to learn fundamental concepts in developmental genetics. In addition, students practice essential skills of scientific communication, including arguing from evidence, developing and testing models, the unique conventions of scientific writing, and the joys of scientific story telling.

biorxiv scientific-communication-and-education 100-200-users 2017

scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells, bioRxiv, 2017-05-18

AbstractParallel single-cell sequencing protocols represent powerful methods for investigating regulatory relationships, including epigenome-transcriptome interactions. Here, we report a novel single-cell method for parallel chromatin accessibility, DNA methylation and transcriptome profiling. scNMT-seq (single-cell nucleosome, methylation and transcription sequencing) uses a GpC methyltransferase to label open chromatin followed by bisulfite and RNA sequencing. We validate scNMT-seq by applying it to differentiating mouse embryonic stem cells, finding links between all three molecular layers and revealing dynamic coupling between epigenomic layers during differentiation.

biorxiv genomics 100-200-users 2017

CRISPR-Cas9 genome editing in human cells works via the Fanconi Anemia pathway, bioRxiv, 2017-05-12

AbstractCRISPR-Cas9 genome editing creates targeted double strand breaks (DSBs) in eukaryotic cells that are processed by cellular DNA repair pathways. Co-administration of single stranded oligonucleotide donor DNA (ssODN) during editing can result in high-efficiency (>20%) incorporation of ssODN sequences into the break site. This process is commonly referred to as homology directed repair (HDR) and here referred to as single stranded template repair (SSTR) to distinguish it from repair using a double stranded DNA donor (dsDonor). The high efficacy of SSTR makes it a promising avenue for the treatment of genetic diseases1,2, but the genetic basis of SSTR editing is still unclear, leaving its use a mostly empiric process. To determine the pathways underlying SSTR in human cells, we developed a coupled knockdown-editing screening system capable of interrogating multiple editing outcomes in the context of thousands of individual gene knockdowns. Unexpectedly, we found that SSTR requires multiple components of the Fanconi Anemia (FA) repair pathway, but does not require Rad51-mediated homologous recombination, distinguishing SSTR from repair using dsDonors. Knockdown of FA genes impacts SSTR without altering break repair by non-homologous end joining (NHEJ) in multiple human cell lines and in neonatal dermal fibroblasts. Our results establish an unanticipated and central role for the FA pathway in templated repair from single stranded DNA by human cells. Therapeutic genome editing has been proposed to treat genetic disorders caused by deficiencies in DNA repair, including Fanconi Anemia. Our data imply that patient genotype andor transcriptome profoundly impact the effectiveness of gene editing treatments and that adjuvant treatments to bias cells towards FA repair pathways could have considerable therapeutic value.

biorxiv cell-biology 100-200-users 2017