Running Cell Ranger count – Songqi Duan

bash

/opt/cellranger/8.0.1/lib/bin/bamtofastq \
  --nthreads=8 \
  --cr11 \
  /datapool/datasets/pbmc3k/downloaded/pbmc3k_possorted_genome_bam.bam \
  /datapool/datasets/pbmc3k/fastqs

bamtofastq v1.4.1

WARNING: no @RG (read group) headers found in BAM file. Splitting data by the GEM well marked in the corrected barcode tag.

Reads without a corrected barcode will not appear in output FASTQs

Unrecognized 10x BAM file.

For BAM files produced by older pipelines, use one of the following flags:

–gemcode BAM files created with GemCode data using Longranger 1.0 - 1.3

–lr20 BAM files created with Longranger 2.0 using Chromium Genome data

–cr11 BAM files created with Cell Ranger 1.0-1.1 using Single Cell 3’ v1 data

bash

/opt/cellranger/8.0.1/bin/cellranger count \
  --id=pbmc3k \
  --transcriptome=/datapool/reference_genomes/gencode/human/47/index/cellranger/GRCh38 \
  --fastqs=/datapool/datasets/pbmc3k/fastqs/gemgroup001 \
  --output-dir=/datapool/datasets/pbmc3k/alignments \
  --localcores=8 \
  --localmem=60 \
  --create-bam true \
  --nosecondary

library(Shennong)
library(Seurat)

Loading required package: SeuratObject

Loading required package: sp

'SeuratObject' was built with package 'Matrix' 1.7.1 but the current
version is 1.7.2; it is recomended that you reinstall 'SeuratObject' as
the ABI for 'Matrix' may have changed


Attaching package: 'SeuratObject'

The following objects are masked from 'package:base':

    intersect, t

library(tidyverse)

── Attaching core tidyverse packages ──────────────────────── tidyverse 2.0.0 ──
✔ dplyr     1.1.4     ✔ readr     2.1.5
✔ forcats   1.0.0     ✔ stringr   1.5.1
✔ ggplot2   3.5.1     ✔ tibble    3.2.1
✔ lubridate 1.9.4     ✔ tidyr     1.3.1
✔ purrr     1.0.4

── Conflicts ────────────────────────────────────────── tidyverse_conflicts() ──
✖ dplyr::filter() masks stats::filter()
✖ dplyr::lag()    masks stats::lag()
ℹ Use the conflicted package (<http://conflicted.r-lib.org/>) to force all conflicts to become errors

outdir <- "/datapool/datasets/pbmc3k/analysis"
dir.create(path = outdir, recursive = TRUE)

Warning in dir.create(path = outdir, recursive = TRUE): cannot create dir
'/datapool', reason 'Permission denied'

counts <- sn_read("https://zenodo.org/records/14868137/files/filtered_feature_bc_matrix.h5?download=1")

seurat_obj <- sn_initialize_seurat_object(
  x = counts, 
  project = "pbmc3k", species = "human"
)

Maps last updated on: Sat Nov 16 10:35:32 2024

Warning in HGNChelper::checkGeneSymbols(x = rownames(counts), species =
species): Human gene symbols should be all upper-case except for the 'orf' in
open reading frames. The case of some letters was corrected.

Warning in HGNChelper::checkGeneSymbols(x = rownames(counts), species =
species): x contains non-approved gene symbols

Warning: Different cells and/or features from existing assay RNA

Maps last updated on: Sat Nov 16 10:35:32 2024
Maps last updated on: Sat Nov 16 10:35:32 2024

Warning in HGNChelper::checkGeneSymbols(genes, species = species): x contains
non-approved gene symbols

seurat_obj <- sn_filter_cells(x = seurat_obj,
        features = c("nFeature_RNA", "nCount_RNA",
                     "percent.mt"))

Warning: Overwriting miscellanous data for qc
Warning: Overwriting miscellanous data for qc

seurat_obj <- seurat_obj |> sn_filter_genes()

seurat_obj <- seurat_obj |> 
  sn_run_cluster()

DimPlot(object = seurat_obj, reduction = "umap", label = TRUE) +
  ggplot2::theme(aspect.ratio = 1)