Single Cell RNA-Seq Analysis of Pancreatic Cells

The pancreas is a complex organ comprised of a diverse set of cell types. Proper function of the pancreas is required to maintain healthy metabolism, and pancreatic dysfunction leads to serious illnesses, including type 1 diabetes. Baron et al performed single cell RNA sequencing in a set of post-mortem human donor pancreatic cells from four subjects and two mouse models to better understand the cellular diversity in the pancreas. Analysis of the data identified previously known cell types as well as rare and novel cell type subpopulations, and created a more detailed characterization of the diversity of those cell types. In this project, we will attempt to replicate their primary findings using current analytical methodology and software packages.

Upon completion of project 4, students will be able to do the following:

  • Process the barcode reads of a single cell sequencing dataset
  • Perform cell-by-gene quantification of UMI counts
  • Perform quality control on a UMI counts matrix
  • Analyze the UMI counts to identify clusters and marker genes for distinct cell type populations
  • Ascribe biological meaning to the clustered cell types and identify novel marker genes associated with them

Note

This project description is much more conceptual than previous projects. This is by design. You are still strongly encouraged to share and discuss strategies with each other and your TAs, and use any materials you find online. Remember: outside of this class, you will be expected to figure out how to solve conceptual problems, often without guidance!

1. Read the paper

Baron, Maayan, Adrian Veres, Samuel L. Wolock, Aubrey L. Faust, Renaud Gaujoux, Amedeo Vetere, Jennifer Hyoje Ryu, et al. 2016. “A Single-Cell Transcriptomic Map of the Human and Mouse Pancreas Reveals Inter- and Intra-Cell Population Structure.” Cell Systems 3 (4): 346–60.e4. PMID: 27667365

2. Process Single Cell Sequencing Reads and Choose Barcodes

Lead role: Data Curator

First we must process the sequencing libraries into UMI counts matrices.

The human single cell RNA-Seq samples in this study have already been downloaded and can be found under /projectnb/bf528/project_4_scrnaseq/fastq. The sequencing protocol used by the paper uses a complicated barcoding scheme for read 1. Typically the barcode for each cell and UMIs for each transcript will be provided to you in a more convenient format, so the raw read 1 barcode has already been processed for you in files with names that look like SRRXXXXXXX_1_bc.fastq.gz. The reads in this file have the format:

BBBBBBBBBBBBBBBBBBBUUUUUU
\   19 bc bases   /\ UMI/

You will need this information when quantifying the reads with salmon. Use these preprocessed files as read 1 instead of the ones ending in _1.fastq.gz.

Note

Some of the barcode reads in the original *_1.fastq.gz could not be matched to the InDrops barcode scheme. This is a common occurence due to noise in the protocol. Furthermore, although the original InDrops barcodes are variable length, the barcodes provided to you above have been padded to be all exactly 19 bases + 6 UMI bases = 25 bases when a valid barcode pattern was identified in the read. Any barcodes that are not exactly 25 bases long will be of very low frequency (probably exactly 1 read per) and therefore will be filtered out when you perform the frequency filter when creating the barcode whitelist in 2.3.

  1. Locate the sample metadata. The sample information corresponding to the short read archives has not been provided to you. Examine the paper for a link to the GEO accession number. On the GEO accession page, search for the SRA Run Selector link at the bottom and identify and download the sample information for the human samples on the linked page. There are 13 samples (sequencing libraries), but only four individuals. Use only the SRR files associated with the 51 year old female donor for further analysis.
  2. Count the number of reads by barcodes. Examine each SRRXXXXXXX_1_bc.fastq.gz and calculate the number of reads per distinct barcode. Hint: Generate a cumulative distribution plot to help understand how the reads are distributed among barcodes. NB: the UMI is not part of the barcode.
  3. Whitelist informative barcodes. We wish to eliminate reads with infrequent barcodes from consideration (why?). Choose a read threshold to filter out barcodes that are too infrequent to be informative and write the remaining distinct barcodes to a file, one barcode per line. These will be our “whitelist” barcodes to provide to salmon in the next step.
  4. Generate UMI counts matrix. Generate UMI matrix using salmon alevin with the provided fastq files and the whitelisted barcodes from the previous step. You will need to provide custom barcode+UMI arguments when quantifying the reads: --end 5 --barcodeLength 19 --umiLength 6. Use the current human reference transcriptome available on the Gencode website. You should also create a transcript ID (ENSTXXX…) to gene (ENSGXXX…) map file as described in the salmon alevin documentation, to enable salmon to collapse from the transcript to the gene level.
  5. Record mapping statistics. The salmon output contains some useful statistics about how well the reads mapped. Capture and examine the output and report any informative statistics (e.g. mapping rate) in your report.

Warning

These files are big and will take a significant amount of time to process. Start early!

Note

Suggestion: you may find it convenient to create smaller versions of the files when developing your code.

Note

Suggestion: salmon can run with multiple threads using the -p argument. You will need to submit your qsub jobs with the appropriate parallel environemt, which you can find on the SCC documentation site

Deliverables:

  • Report and justify the strategy you chose for identifying informative barcodes by whatever method you deem appropriate
  • Report any mapping statistics from the salmon output you deem relevant
  • UMI counts matrix for the chosen sample in the form of salmon alevin output

3. Processing the UMI counts matrix

Lead role: Programmer

The authors developed a custom analysis methodology for this paper. We will follow the tutorial materials written by the authors of the Bioconductor package Seurat instead of trying to implement their methods.

A precomputed UMI counts matrix is available at

/projectnb/bf528/project_4_scrnaseq/GSM2230760__salmon_quant

for your use. Hint: you may find this tutorial instructive in completing many of these steps.

  1. Filter out low-quality cells. You will need to discover how to load the salmon alevin counts file into R. Choose criteria to identify and filter low-quality cells and genes from the UMI counts matrix. You should consider the minimum number of non-zero count genes per cell, the number of non-zero count cells per gene, etc. NB: The UMI counts matrix has Ensembl gene identifiers, but it may be helpful to map them to gene symbols. There are many ways to do this.
  2. Filter out low variance genes. Further filter the counts matrix to include only highly variable features that are likely to be informative. Choose an appropriate number of features based on the data. Recall that the counts matrix must be normalized before comparing cells to one another.
  3. Identify clusters of cell type subpopulations. Use a clustering method of your choosing to discover clusters of cells. Identify the number of cells in each cluster.

Deliverables:

  • Report the number of cells and the number of genes in the unfiltered dataset
  • Report the number of cells remaining after filtering out low quality cells, and justify any filtering criteria used
  • Report the number features you used after variance filtering, and justify the filtering criteria used
  • Report the number of clusters you identified, and report the relative proportions of cell numbers (e.g. as a bar or pie chart)

4. Cluster marker genes

Lead role: Analyst

The authors report that they found all major pancreatic cell types in the data based on known marker genes. The major cell types also had approximately expected relative proportions.

A RDS file containing a saved Seurat object with processed, clustered counts is available for you to use:

/projectnb/bf528/project_4_scrnaseq/GSM2230760_seurat.rda

You can load this into R using cells <- loadRDS("GSM2230760_seurat.rda"). Hint: you may find this tutorial instructive in completing many of these steps.

  1. Identify marker genes for each cluster. Use a differential expression method to identify the marker genes in each cluster. You may use any reasonable approach to do this.
  2. Label clusters as a cell type based on marker genes. The marker genes should indicate which clusters correspond to which cell types. You may use the marker genes indicated by the authors or do your own research.
  3. Visualize the clustered cells using a projection method. Use either t-SNE or UMAP to create a projection and scatter plot colored by cell cluster. Label the plot with the cell types you previously assigned. Compare this plot to Figure 1D in the paper.
  4. Visualize the top marker genes per cluster. Choose however many top marker genes you deem suitable and visualize them to show the differences. You might plot a clustered heatmap of log normalized UMI counts for those genes across all cells following the format used in Figure 1B of the paper or any other format you deem effective. The cluster identities should be clearly visible on the heatmap, probably as a colored bar along one of the axes.
  5. Find novel marker genes. After labeling your clusters, there will likely be other differentially expressed genes in that cluster that are just as discriminative of cell type as the marker genes. Indicate these genes in your report by whatever means you deem appropriate.
  6. Four member groups only: Output marker gene information. Write out a CSV file with marker gene statistics you identified above. You should include the fold change, precision (i.e. p-value and adjusted p-value), cluster membership, and gene identifier. The Biologist will use these marker genes for further analysis.

Deliverables:

  • Report the method you used to label clusters to cell types
  • Compare your cluster marker genes to those reported in the paper; do they agree? Are there clusters that could not be labeled using their marker genes? What do these clusters seem to be?
  • Show the visualized projection plot colored by cluster and labeled with your assigned cell type
  • Compare your projection plot with that of the paper in your discussion
  • Show the clustered heatmap of log normalized UMI counts for marker genes of each cluster
  • Compare your heatmap results with that of the paper in your discussion
  • Report any “novel” marker genes in an appropriate format, e.g. a table or in the heatmap

5. In-depth marker gene analysis

Lead role: Biologist - for 4 person groups only

The authors labeled their clusters to mostly previously known cell types that are thought to perform certain functions. We will attempt to confirm these cell type functions using the marker genes that were discovered through the relatively unbiased clustering and differential expression analysis, without the influence of a priori knowledge.

A precomputed clustered dataset is available for you to use at:

/projectnb/bf528/project_4_scrnaseq/GSM2230760_marker_genes.csv
  1. Perform gene set enrichment analysis on marker genes. Use an appropriate gene set enrichment analysis tool of your choosing (e.g. DAVID, enrichR, metascape, etc) to perform gene set enrichment on the marker genes for each cluster. You might decide to filter the marker genes by some criteria, like adjusted p-value or log2 fold change and see how the results change. Report the results and compare them to the cell type label, if any, of each cluster.

Deliverables:

  • Report the results of the gene set enrichment analysis in a concise way (e.g. a table with cluster name, label, number of marker genes, and enriched gene sets, ensuring the table is readable and not too large)
  • Discuss the results in the Discussion section, describing how well you think the gene set enrichment results match the assigned cell type labels

8. Discuss Your Findings

Discuss your findings with your team members and other teams. Some interesting questions to consider:

  1. Did you replicate the study? What were they differences between their methodology and those you performed?
  2. Were there any key differences between your results and those presented in the study? Did these key differences affect the biological interpretation of the results?
  3. Did you find evidence of the distinct cell types described in the paper? Were there any major differences in the cell types you discovered, e.g. in number, proportion, markers, etc?