HSSHMP_2024

Code for reproducing the figures and results in the preprint Accurate quantification of single-nucleus and single-cell RNA-seq transcripts by Kristján Eldjárn Hjörleifsson, Delaney Sullivan, Nikhila Swarna, Guillaume Holley, Páll Melsted and Lior Pachter

Note about human reference genome

The human reference genome (FASTA+GTF) used in all analyses is available directly at https://github.com/pachterlab/HSSHMP_2024/releases under the filename human_CR_3.0.0.tar.gz.

Introduction

Please follow the steps below in order to reproduce the results of the preprint. Set all the paths to be relative to the directory HSHMP_2022.

main_path="$(pwd)/HSSHMP_2024"
kallisto="$main_path/kallisto_0.48.0/kallisto"
kallisto="$main_path/kallisto_0.50.0/kallisto"
kallisto="$main_path/kallisto_0.50.1/kallisto"
bustools="$main_path/bustools/build/src/bustools"
cellranger7="$main_path/cellranger/cellranger-7.0.1/cellranger"
salmon="$main_path/salmon-latest_linux_x86_64/bin/salmon"

Download software

kallisto

version 0.48.0

cd $main_path
wget https://github.com/pachterlab/kallisto/releases/download/v0.48.0/kallisto_linux-v0.48.0.tar.gz
tar -xzvf kallisto_linux-v0.48.0.tar.gz
mv kallisto kallisto_0.48.0

version 0.50.0

cd $main_path
wget https://github.com/pachterlab/kallisto/releases/download/v0.50.0/kallisto_linux-v0.50.0.tar.gz
tar -xzvf kallisto_linux-v0.50.0.tar.gz
mv kallisto kallisto_0.50.0

version 0.50.1

cd $main_path
wget https://github.com/pachterlab/kallisto/releases/download/v0.50.1/kallisto_linux-v0.50.1.tar.gz
tar -xzvf kallisto_linux-v0.50.1.tar.gz
mv kallisto kallisto_0.50.1

bustools

version 0.43.2

cd $main_path
rm -rf bustools
git clone -b v0.43.2 https://github.com/BUStools/bustools
cd bustools && mkdir -p build && cd build
cmake .. && make

kb-python

version 0.28.0

cd $main_path
yes|python -m pip uninstall kb-python
python -m pip install kb_python==0.28.0

Cell Ranger

Note: Cell Ranger needs to be installed manually. Version is as follows:

Cell Ranger v7.0.1 (Released August 18, 2022. Downloaded October 7, 2022)

salmon-alevin-fry

salmon version 1.10.0; alevin-fry version 0.8.2; pyroe 0.9.3; simpleaf 0.15.1

cd $main_path
wget https://github.com/COMBINE-lab/salmon/releases/download/v1.10.0/salmon-1.10.0_linux_x86_64.tar.gz && tar -xzvf salmon-1.10.0_linux_x86_64.tar.gz
export RUSTUP_HOME=${main_path}/.rustup/
export CARGO_HOME=${main_path}/.cargo/
curl --proto '=https' --tlsv1.2 -sSf https://sh.rustup.rs | sh
./.cargo/bin/cargo install --version 0.8.2 --force alevin-fry
./.cargo/bin/cargo install --version 0.15.1 --force simpleaf
yes|python -m pip uninstall pyroe
python -m pip install pyroe==0.9.3

simpleaf configuration:

export ALEVIN_FRY_HOME="$main_path/af_home"
simpleaf set-paths \
--salmon $(pwd)/salmon-latest_linux_x86_64/bin/salmon

simpleaf workflow get --name 10x-chromium-3p-v3 -o af10xv3

cellCounts

Open up an R session and then run:

if (!require("BiocManager", quietly = TRUE))
    install.packages("BiocManager")

BiocManager::install("Rsubread")

Note: Version Rsubread_2.12.3

STARSolo simulations

Navigate to STARsoloManuscript and run the scripts there

Note: Make sure to run the STARsoloManuscript scripts first before proceeding (we use these indices and the links to the program binary files downstream). At a minimum, complete the sections "Create symlinks to executables", "Create indices", and "Mouse genome prep".

Human datasets

Single-cell

wget https://s3-us-west-2.amazonaws.com/10x.files/samples/cell-exp/6.1.0/20k_PBMC_3p_HT_nextgem_Chromium_X/20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs.tar
tar -xvf 20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs.tar

Single-nucleus

wget https://cf.10xgenomics.com/samples/cell-exp/7.0.0/5k_human_jejunum_CNIK_3pv3/5k_human_jejunum_CNIK_3pv3_fastqs.tar
tar -xvf 5k_human_jejunum_CNIK_3pv3_fastqs.tar

Mouse datasets

Single-cell

wget https://s3-us-west-2.amazonaws.com/10x.files/samples/cell-exp/4.0.0/SC3_v3_NextGem_SI_Neuron_10K/SC3_v3_NextGem_SI_Neuron_10K_fastqs.tar
tar -xvf SC3_v3_NextGem_SI_Neuron_10K_fastqs.tar

Single-nucleus

wget https://s3-us-west-2.amazonaws.com/10x.files/samples/cell-exp/7.0.0/5k_mouse_lung_CNIK_3pv3/5k_mouse_lung_CNIK_3pv3_fastqs.tar
tar -xvf 5k_mouse_lung_CNIK_3pv3_fastqs.tar

Spatial

wget https://s3-us-west-2.amazonaws.com/10x.files/samples/spatial-exp/2.1.0/CytAssist_11mm_FFPE_Mouse_Embryo/CytAssist_11mm_FFPE_Mouse_Embryo_fastqs.tar
tar -xf CytAssist_11mm_FFPE_Mouse_Embryo_fastqs.tar && rm CytAssist_11mm_FFPE_Mouse_Embryo_fastqs.tar && mv fastqs/* ./ && rmdir fastqs

Generate count matrix for datasets using kallisto

kb count --kallisto STARsoloManuscript/exe/kallisto_0.50.1 --bustools STARsoloManuscript/exe/bustools_0.43.2 -t 20 -x 10XV3 \
    --workflow nac --sum=total -i STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_offlist_1/index.idx \
    -g STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_offlist_1/g \
    -c1 STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_offlist_1/c1 \
    -c2 STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_offlist_1/c2 \
    -o ./matrices_human_20k_PBMC/ --overwrite --verbose \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L001_R1_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L001_R2_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L002_R1_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L002_R2_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L003_R1_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L003_R2_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L004_R1_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L004_R2_001.fastq.gz

kb count --kallisto STARsoloManuscript/exe/kallisto_0.50.1 --bustools STARsoloManuscript/exe/bustools_0.43.2 -t 20 -x 10XV3 \
    --workflow nac --sum=total -i STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_offlist_1/index.idx \
    -g STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_offlist_1/g \
    -c1 STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_offlist_1/c1 \
    -c2 STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_offlist_1/c2 \
    -o ./matrices_human_5k_jejunum_nuclei/ --overwrite --verbose \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L001_R1_001.fastq.gz \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L001_R2_001.fastq.gz \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L002_R1_001.fastq.gz \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L002_R2_001.fastq.gz \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L003_R1_001.fastq.gz \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L003_R2_001.fastq.gz \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L004_R1_001.fastq.gz \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L004_R2_001.fastq.gz

kb count --kallisto STARsoloManuscript/exe/kallisto_0.50.1 --bustools STARsoloManuscript/exe/bustools_0.43.2 -t 20 -x 10XV3 \
    --workflow nac --sum=total -i STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_offlist_1/index.idx \
    -g STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_offlist_1/g \
    -c1 STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_offlist_1/c1 \
    -c2 STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_offlist_1/c2 \
    -o ./matrices_mouse_10k_neuron/ --overwrite --verbose \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L002_R1_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L002_R2_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L003_R1_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L003_R2_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L004_R1_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L004_R2_001.fastq.gz

kb count --kallisto STARsoloManuscript/exe/kallisto_0.50.1 --bustools STARsoloManuscript/exe/bustools_0.43.2 -t 20 -x 10XV3 \
    --workflow nac --sum=total -i STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_offlist_1/index.idx \
    -g STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_offlist_1/g \
    -c1 STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_offlist_1/c1 \
    -c2 STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_offlist_1/c2 \
    -o ./matrices_mouse_5k_lung/ --overwrite --verbose \
    5k_mouse_lung_CNIK_3pv3_fastqs/5k_mouse_lung_CNIK_3pv3_S4_L001_R1_001.fastq.gz \
    5k_mouse_lung_CNIK_3pv3_fastqs/5k_mouse_lung_CNIK_3pv3_S4_L001_R2_001.fastq.gz \
    5k_mouse_lung_CNIK_3pv3_fastqs/5k_mouse_lung_CNIK_3pv3_S4_L002_R1_001.fastq.gz \
    5k_mouse_lung_CNIK_3pv3_fastqs/5k_mouse_lung_CNIK_3pv3_S4_L002_R2_001.fastq.gz \
    5k_mouse_lung_CNIK_3pv3_fastqs/5k_mouse_lung_CNIK_3pv3_S4_L003_R1_001.fastq.gz \
    5k_mouse_lung_CNIK_3pv3_fastqs/5k_mouse_lung_CNIK_3pv3_S4_L003_R2_001.fastq.gz

kb count --kallisto STARsoloManuscript/exe/kallisto_0.50.1 --bustools STARsoloManuscript/exe/bustools_0.43.2 -t 20 -x VISIUM \
    --strand=unstranded --workflow nac --sum=total -i STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_offlist_1/index.idx \
    -g STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_offlist_1/g \
    -c1 STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_offlist_1/c1 \
    -c2 STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_offlist_1/c2 \
    -o ./matrices_mouse_ffpe/ --overwrite --verbose \
    CytAssist_11mm_FFPE_Mouse_Embryo_fastqs/CytAssist_11mm_FFPE_Mouse_Embryo_S1_L004_R1_001.fastq.gz \
    CytAssist_11mm_FFPE_Mouse_Embryo_fastqs/CytAssist_11mm_FFPE_Mouse_Embryo_S1_L004_R2_001.fastq.gz

Filtering for UMI threshold >= 500 (applies to total count matrix; the other count matrices just use the barcodes from the "total" matrix).

./filter.sh matrices_human_20k_PBMC 500
./filter.sh matrices_human_5k_jejunum_nuclei 500
./filter.sh matrices_mouse_10k_neuron 500
./filter.sh matrices_mouse_5k_lung 500
./filter.sh matrices_mouse_ffpe 500

Let's now use the script from the simulations (where we compared output matrix vs simulated truth matrix) to now compare our nascent/mature/ambiguous/etc. matrices. Everything is in the mtx_comparisons.sh file.

./mtx_comparisons.sh matrices_human_20k_PBMC
./mtx_comparisons.sh matrices_human_5k_jejunum_nuclei
./mtx_comparisons.sh matrices_mouse_10k_neuron
./mtx_comparisons.sh matrices_mouse_5k_lung
./mtx_comparisons.sh matrices_mouse_ffpe

The final analysis is produced in the matrix_comparisons.ipynb python notebook file.

Runtime and memory benchmarks of kallisto

Note: kb-python already uses the 10xv3 prepackaged on-list.

Note: After the following commands are run, the analysis_dlist_performance.ipynb python notebook contains the final plots.

mkdir -p performance_comparisons/

Human 20k pbmc

nac + offlist:

cmd1="kb count --kallisto STARsoloManuscript/exe/kallisto_0.50.1 --bustools STARsoloManuscript/exe/bustools_0.43.2 -t "
cmd2=" -x 10XV3 \
    --workflow nac -i STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_offlist_1/index.idx \
    -g STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_offlist_1/g \
    -c1 STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_offlist_1/c1 \
    -c2 STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_offlist_1/c2 \
    -o ./performance_comparisons/tmp/ --overwrite \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L001_R1_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L001_R2_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L002_R1_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L002_R2_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L003_R1_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L003_R2_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L004_R1_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L004_R2_001.fastq.gz"

/usr/bin/time -v $cmd1 16 $cmd2 2> performance_comparisons/16_nac_offlist-20kb_PBMC_1.txt

nac (no offlist):

cmd1="kb count --kallisto STARsoloManuscript/exe/kallisto_0.50.1 --bustools STARsoloManuscript/exe/bustools_0.43.2 -t "
cmd2=" -x 10XV3 \
    --workflow nac -i STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_1/index.idx \
    -g STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_1/g \
    -c1 STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_1/c1 \
    -c2 STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_1/c2 \
    -o ./performance_comparisons/tmp/ --overwrite \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L001_R1_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L001_R2_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L002_R1_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L002_R2_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L003_R1_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L003_R2_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L004_R1_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L004_R2_001.fastq.gz"

/usr/bin/time -v $cmd1 16 $cmd2 2> performance_comparisons/16_nac-20kb_PBMC_1.txt

standard + offlist:

cmd1="kb count --kallisto STARsoloManuscript/exe/kallisto_0.50.1 --bustools STARsoloManuscript/exe/bustools_0.43.2 -t "
cmd2=" -x 10XV3 \
    --workflow standard -i STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/standard_offlist_1/index.idx \
    -g STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/standard_offlist_1/g \
    -o ./performance_comparisons/tmp/ --overwrite \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L001_R1_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L001_R2_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L002_R1_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L002_R2_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L003_R1_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L003_R2_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L004_R1_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L004_R2_001.fastq.gz"

/usr/bin/time -v $cmd1 16 $cmd2 2> performance_comparisons/16_standard_offlist-20kb_PBMC_1.txt

standard (no offlist):

cmd1="kb count --kallisto STARsoloManuscript/exe/kallisto_0.50.1 --bustools STARsoloManuscript/exe/bustools_0.43.2 -t "
cmd2=" -x 10XV3 \
    --workflow standard -i STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/standard_1/index.idx \
    -g STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/standard_1/g \
    -o ./performance_comparisons/tmp/ --overwrite \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L001_R1_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L001_R2_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L002_R1_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L002_R2_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L003_R1_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L003_R2_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L004_R1_001.fastq.gz \
    20k_PBMC_3p_HT_nextgem_Chromium_X_fastqs/20k_PBMC_3p_HT_nextgem_Chromium_X_S3_L004_R2_001.fastq.gz"

/usr/bin/time -v $cmd1 16 $cmd2 2> performance_comparisons/16_standard-20kb_PBMC_1.txt

Human 5k jejunum

nac + offlist:

cmd1="kb count --kallisto STARsoloManuscript/exe/kallisto_0.50.1 --bustools STARsoloManuscript/exe/bustools_0.43.2 -t "
cmd2=" -x 10XV3 \
    --workflow nac -i STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_offlist_1/index.idx \
    -g STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_offlist_1/g \
    -c1 STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_offlist_1/c1 \
    -c2 STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_offlist_1/c2 \
    -o ./performance_comparisons/tmp/ --overwrite \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L001_R1_001.fastq.gz \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L001_R2_001.fastq.gz \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L002_R1_001.fastq.gz \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L002_R2_001.fastq.gz \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L003_R1_001.fastq.gz \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L003_R2_001.fastq.gz \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L004_R1_001.fastq.gz \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L004_R2_001.fastq.gz"

/usr/bin/time -v $cmd1 16 $cmd2 2> performance_comparisons/16_nac_offlist-5kb_jejunum_1.txt

nac (no offlist):

cmd1="kb count --kallisto STARsoloManuscript/exe/kallisto_0.50.1 --bustools STARsoloManuscript/exe/bustools_0.43.2 -t "
cmd2=" -x 10XV3 \
    --workflow nac -i STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_1/index.idx \
    -g STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_1/g \
    -c1 STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_1/c1 \
    -c2 STARsoloManuscript/genomes/index/kallisto_0.50.1/human_CR_3.0.0/nac_1/c2 \
    -o ./performance_comparisons/tmp/ --overwrite \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L001_R1_001.fastq.gz \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L001_R2_001.fastq.gz \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L002_R1_001.fastq.gz \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L002_R2_001.fastq.gz \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L003_R1_001.fastq.gz \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L003_R2_001.fastq.gz \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L004_R1_001.fastq.gz \
    5k_human_jejunum_CNIK_3pv3_fastqs/5k_human_jejunum_CNIK_3pv3_S1_L004_R2_001.fastq.gz"

/usr/bin/time -v $cmd1 16 $cmd2 2> performance_comparisons/16_nac-5kb_jejunum_1.txt

Mouse 10k neuron

nac + offlist:

cmd1="kb count --kallisto STARsoloManuscript/exe/kallisto_0.50.1 --bustools STARsoloManuscript/exe/bustools_0.43.2 -t "
cmd2=" -x 10XV3 \
    --workflow nac -i STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_offlist_1/index.idx \
    -g STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_offlist_1/g \
    -c1 STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_offlist_1/c1 \
    -c2 STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_offlist_1/c2 \
    -o ./performance_comparisons/tmp/ --overwrite \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L002_R1_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L002_R2_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L003_R1_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L003_R2_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L004_R1_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L004_R2_001.fastq.gz"

/usr/bin/time -v $cmd1 16 $cmd2 2> performance_comparisons/16_nac_offlist-10kb_neuron_1.txt

nac (no offlist):

cmd1="kb count --kallisto STARsoloManuscript/exe/kallisto_0.50.1 --bustools STARsoloManuscript/exe/bustools_0.43.2 -t "
cmd2=" -x 10XV3 \
    --workflow nac -i STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_1/index.idx \
    -g STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_1/g \
    -c1 STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_1/c1 \
    -c2 STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_1/c2 \
    -o ./performance_comparisons/tmp/ --overwrite \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L002_R1_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L002_R2_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L003_R1_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L003_R2_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L004_R1_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L004_R2_001.fastq.gz"

/usr/bin/time -v $cmd1 16 $cmd2 2> performance_comparisons/16_nac-10kb_neuron_1.txt

standard + offlist:

cmd1="kb count --kallisto STARsoloManuscript/exe/kallisto_0.50.1 --bustools STARsoloManuscript/exe/bustools_0.43.2 -t "
cmd2=" -x 10XV3 \
    --workflow standard -i STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/standard_offlist_1/index.idx \
    -g STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/standard_offlist_1/g \
    -o ./performance_comparisons/tmp/ --overwrite \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L002_R1_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L002_R2_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L003_R1_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L003_R2_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L004_R1_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L004_R2_001.fastq.gz"

/usr/bin/time -v $cmd1 16 $cmd2 2> performance_comparisons/16_standard_offlist-10kb_neuron_1.txt

standard (no offlist):

cmd1="kb count --kallisto STARsoloManuscript/exe/kallisto_0.50.1 --bustools STARsoloManuscript/exe/bustools_0.43.2 -t "
cmd2=" -x 10XV3 \
    --workflow standard -i STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/standard_1/index.idx \
    -g STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/standard_1/g \
    -o ./performance_comparisons/tmp/ --overwrite \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L002_R1_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L002_R2_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L003_R1_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L003_R2_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L004_R1_001.fastq.gz \
    SC3_v3_NextGem_SI_Neuron_10K_fastqs/SC3_v3_NextGem_SI_Neuron_10K_S1_L004_R2_001.fastq.gz"

/usr/bin/time -v $cmd1 16 $cmd2 2> performance_comparisons/16_standard-10kb_neuron_1.txt

Mouse 5k lung

nac + offlist:

cmd1="kb count --kallisto STARsoloManuscript/exe/kallisto_0.50.1 --bustools STARsoloManuscript/exe/bustools_0.43.2 -t "
cmd2=" -x 10XV3 \
    --workflow nac -i STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_offlist_1/index.idx \
    -g STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_offlist_1/g \
    -c1 STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_offlist_1/c1 \
    -c2 STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_offlist_1/c2 \
    -o ./performance_comparisons/tmp/ --overwrite \
    5k_mouse_lung_CNIK_3pv3_fastqs/5k_mouse_lung_CNIK_3pv3_S4_L001_R1_001.fastq.gz \
    5k_mouse_lung_CNIK_3pv3_fastqs/5k_mouse_lung_CNIK_3pv3_S4_L001_R2_001.fastq.gz \
    5k_mouse_lung_CNIK_3pv3_fastqs/5k_mouse_lung_CNIK_3pv3_S4_L002_R1_001.fastq.gz \
    5k_mouse_lung_CNIK_3pv3_fastqs/5k_mouse_lung_CNIK_3pv3_S4_L002_R2_001.fastq.gz \
    5k_mouse_lung_CNIK_3pv3_fastqs/5k_mouse_lung_CNIK_3pv3_S4_L003_R1_001.fastq.gz \
    5k_mouse_lung_CNIK_3pv3_fastqs/5k_mouse_lung_CNIK_3pv3_S4_L003_R2_001.fastq.gz"

/usr/bin/time -v $cmd1 16 $cmd2 2> performance_comparisons/16_nac_offlist-5kb_lung_1.txt

nac (no offlist):

cmd1="kb count --kallisto STARsoloManuscript/exe/kallisto_0.50.1 --bustools STARsoloManuscript/exe/bustools_0.43.2 -t "
cmd2=" -x 10XV3 \
    --workflow nac -i STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_1/index.idx \
    -g STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_1/g \
    -c1 STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_1/c1 \
    -c2 STARsoloManuscript/genomes/index/kallisto_0.50.1/mouse/nac_1/c2 \
    -o ./performance_comparisons/tmp/ --overwrite \
    5k_mouse_lung_CNIK_3pv3_fastqs/5k_mouse_lung_CNIK_3pv3_S4_L001_R1_001.fastq.gz \
    5k_mouse_lung_CNIK_3pv3_fastqs/5k_mouse_lung_CNIK_3pv3_S4_L001_R2_001.fastq.gz \
    5k_mouse_lung_CNIK_3pv3_fastqs/5k_mouse_lung_CNIK_3pv3_S4_L002_R1_001.fastq.gz \
    5k_mouse_lung_CNIK_3pv3_fastqs/5k_mouse_lung_CNIK_3pv3_S4_L002_R2_001.fastq.gz \
    5k_mouse_lung_CNIK_3pv3_fastqs/5k_mouse_lung_CNIK_3pv3_S4_L003_R1_001.fastq.gz \
    5k_mouse_lung_CNIK_3pv3_fastqs/5k_mouse_lung_CNIK_3pv3_S4_L003_R2_001.fastq.gz"

/usr/bin/time -v $cmd1 16 $cmd2 2> performance_comparisons/16_nac-5kb_lung_1.txt

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HSSHMP_2024

Note about human reference genome

Introduction

Download software

kallisto

bustools

kb-python

Cell Ranger

salmon-alevin-fry

cellCounts

STARSolo simulations

Human datasets

Single-cell

Single-nucleus

Mouse datasets

Single-cell

Single-nucleus

Spatial

Generate count matrix for datasets using kallisto

Runtime and memory benchmarks of kallisto

Human 20k pbmc

Human 5k jejunum

Mouse 10k neuron

Mouse 5k lung

About

Releases 1

Packages

Contributors 2

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Name		Name	Last commit message	Last commit date
Latest commit History 4 Commits
STARsoloManuscript		STARsoloManuscript
matrices_human_20k_PBMC		matrices_human_20k_PBMC
matrices_human_5k_jejunum_nuclei		matrices_human_5k_jejunum_nuclei
matrices_mouse_10k_neuron		matrices_mouse_10k_neuron
matrices_mouse_5k_lung		matrices_mouse_5k_lung
matrices_mouse_ffpe		matrices_mouse_ffpe
performance_comparisons		performance_comparisons
LICENSE		LICENSE
README.md		README.md
analysis_dlist_performance.ipynb		analysis_dlist_performance.ipynb
do_sum_matrices.py		do_sum_matrices.py
filter.sh		filter.sh
matrix_comparisons.ipynb		matrix_comparisons.ipynb
mtx_comparisons.sh		mtx_comparisons.sh
pseudobulk.py		pseudobulk.py

License

pachterlab/HSSHMP_2024

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Latest commit

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Repository files navigation

HSSHMP_2024

Note about human reference genome

Introduction

Download software

kallisto

bustools

kb-python

Cell Ranger

salmon-alevin-fry

cellCounts

STARSolo simulations

Human datasets

Single-cell

Single-nucleus

Mouse datasets

Single-cell

Single-nucleus

Spatial

Generate count matrix for datasets using kallisto

Runtime and memory benchmarks of kallisto

Human 20k pbmc

Human 5k jejunum

Mouse 10k neuron

Mouse 5k lung

About

Resources

License

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Releases 1

Packages 0

Contributors 2

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Packages