CellRanger on Ehrens scRNAseq data

Background

This post describes the initial QC analysis for scRNAseq on Acropora cervicornis and Pocillopora damicornis using the new 10X GEMX cel capture technology. FYI to analyze this data, you must use cellranger/8.0.0.
For each species, there are two separate libraries: 001 = All cells, 002 = ALDH+ cells

Acropora cervicornis analysis

Downloading Acropora cervicornis genome

https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_032359415.1/

scp -r GCA_032359415.1.zip kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/

unzip GCA_032359415.1.zip

I need to convert the .fna file to .fa for STAR to work properly

cd GCA_032359415.1
cp GCA_032359415.1_NEU_Acer_K2_genomic.fna GCA_032359415.1_NEU_Acer_K2_genomic.fa

Make reference

nano mkref2.job

#!/bin/bash

#BSUB -J scSeq_mkref_acer
#BSUB -q general
#BSUB -P dark_genes
#BSUB -n 6
#BSUB -W 12:00
#BSUB -u kxw755@earth.miami.edu
#BSUB -o acer_mkref_2.out
#BSUB -e acer_mkref_2.err

###################################################################

module load cellranger/8.0.0

cellranger mkref \
--genome=acer_K2_2 --fasta=GCA_032359415.1_NEU_Acer_K2_genomic.fa --genes=genomic.gtf

bsub < mkref2.job

/nethome/kxw755/GCA_032359415.1/acer_K2

Count

Ehrens-18003-001 library (All Cells)

cd /nethome/kxw755/20240418_Ehrens-18003-001_GEX3_GEMX

nano count_001.job

#BSUB -J count_18003-001
#BSUB -q bigmem
#BSUB -P dark_genes
#BSUB -n 16
#BSUB -W 120:00
#BSUB -R "rusage[mem=15000]"
#BSUB -u kxw755@earth.miami.edu
#BSUB -o count_18003-001.out
#BSUB -e count_18003-001.err
#BSUB -B
#BSUB -N
###################################################################

module load cellranger/8.0.0

cellranger count \
 --id=18003-001 \
 --transcriptome=/nethome/kxw755/GCA_032359415.1/acer_K2_2   \
 --fastqs=/nethome/kxw755/20240418_Ehrens-18003-001_GEX3_GEMX \
 --sample=Ehrens-18003-001_GEX3_GEMX \
 --create-bam=true

bsub < count_001.job

Export

scp -r kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/20240418_Ehrens-18003-001_GEX3_GEMX/18003-001/outs/web_summary.html /Users/kxw755/Desktop/Ehrens_StemCells/20240418_Ehrens-18003-001_web_summary.html

scp -r kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/20240418_Ehrens-18003-001_GEX3_GEMX/18003-001/outs/filtered_feature_bc_matrix.h5 /Users/kxw755/Desktop/Ehrens_StemCells/20240418_Ehrens-18003-001_filtered_feature_bc_matrix.h5

scp -r kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/20240418_Ehrens-18003-001_GEX3_GEMX/18003-001/outs/metrics_summary.csv /Users/kxw755/Desktop/Ehrens_StemCells/20240418_Ehrens-18003-001_metrics_summary.csv

Ehrens-18003-002 library (ALDH+)

cd /nethome/kxw755/20240418_Ehrens-18003-002_GEX3_GEMX

nano count_002.job

#BSUB -J count_18003-002
#BSUB -q bigmem
#BSUB -P dark_genes
#BSUB -n 16
#BSUB -W 120:00
#BSUB -R "rusage[mem=15000]"
#BSUB -u kxw755@earth.miami.edu
#BSUB -o count_18003-002.out
#BSUB -e count_18003-002.err
#BSUB -B
#BSUB -N
###################################################################

module load cellranger/8.0.0

cellranger count \
 --id=18003-002 \
 --transcriptome=/nethome/kxw755/GCA_032359415.1/acer_K2_2 \
 --fastqs=/nethome/kxw755/20240418_Ehrens-18003-002_GEX3_GEMX \
 --sample=Ehrens-18003-002_GEX3_GEMX \
 --create-bam=true

bsub < count_002.job

Export

scp -r kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/20240418_Ehrens-18003-002_GEX3_GEMX/18003-002/outs/web_summary.html /Users/kxw755/Desktop/Ehrens_StemCells/20240418_Ehrens-18003-002_web_summary.html

scp -r kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/20240418_Ehrens-18003-002_GEX3_GEMX/18003-002/outs/filtered_feature_bc_matrix.h5 /Users/kxw755/Desktop/Ehrens_StemCells/20240418_Ehrens-18003-002_filtered_feature_bc_matrix.h5

scp -r kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/20240418_Ehrens-18003-002_GEX3_GEMX/18003-002/outs/metrics_summary.csv /Users/kxw755/Desktop/Ehrens_StemCells/20240418_Ehrens-18003-002_metrics_summary.csv

Pocillopora damicornis analysis

Download Pocillopora damicornis genome

https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_003704095.1/

scp -r pdam_genome kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/

I need to convert the .fna file to .fa for STAR to work properly

cd GCA_032359415.1
cp GCA_003704095.1_ASM370409v1_genomic.fna GCA_003704095.1_ASM370409v1_genomic.fa

Make reference

nano mkref.job

#!/bin/bash

#BSUB -J scSeq_mkref_pdam
#BSUB -q general
#BSUB -P dark_genes
#BSUB -n 6
#BSUB -W 12:00
#BSUB -u kxw755@earth.miami.edu
#BSUB -o pdam_mkref_2.out
#BSUB -e pdam_mkref_2.err
#BSUB -B
#BSUB -N

###################################################################

module load cellranger/8.0.0

cellranger mkref \
--genome=pdam --fasta=GCA_003704095.1_ASM370409v1_genomic.fa --genes=genomic.gtf

bsub < mkref.job

Count

Ehrens-18114-001 library (All Cells)

cd /nethome/kxw755/20240501_Ehrens-18114-001_GEX3_GEMX

nano count_001.job

#BSUB -J count_18114-001
#BSUB -q bigmem
#BSUB -P dark_genes
#BSUB -n 16
#BSUB -W 120:00
#BSUB -R "rusage[mem=15000]"
#BSUB -u kxw755@earth.miami.edu
#BSUB -o count_18114-001.out
#BSUB -e count_18114-001.err
#BSUB -B
#BSUB -N
###################################################################

module load cellranger/8.0.0

cellranger count \
 --id=18114-001 \
 --transcriptome=/nethome/kxw755/pdam_genome/pdam   \
 --fastqs=/nethome/kxw755/20240501_Ehrens-18114-001_GEX3_GEMX \
 --sample=Ehrens-18114-001_GEX3_GEMX \
 --create-bam=false

bsub < count_001.job

Export

scp -r kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/20240501_Ehrens-18114-001_GEX3_GEMX/18114-001/outs/web_summary.html /Users/kxw755/Desktop/Ehrens_StemCells/20240501_Ehrens-18114-001_web_summary.html

scp -r kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/20240501_Ehrens-18114-001_GEX3_GEMX/18114-001/outs/filtered_feature_bc_matrix.h5 /Users/kxw755/Desktop/Ehrens_StemCells/20240501_Ehrens-18114-001_filtered_feature_bc_matrix.h5

scp -r kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/20240501_Ehrens-18114-001_GEX3_GEMX/18114-001/outs/metrics_summary.csv /Users/kxw755/Desktop/Ehrens_StemCells/20240501_Ehrens-18114-001_metrics_summary.csv

Ehrens-18114-002 library (ALDH+)

cd /nethome/kxw755/20240501_Ehrens-18114-002_GEX3_GEMX

nano count_002.job

#BSUB -J count_18114-002
#BSUB -q bigmem
#BSUB -P dark_genes
#BSUB -n 16
#BSUB -W 120:00
#BSUB -R "rusage[mem=15000]"
#BSUB -u kxw755@earth.miami.edu
#BSUB -o count_18114-002.out
#BSUB -e count_18114-002.err
#BSUB -B
#BSUB -N
###################################################################

module load cellranger/8.0.0

cellranger count \
 --id=18114-002 \
 --transcriptome=/nethome/kxw755/pdam_genome/pdam   \
 --fastqs=/nethome/kxw755/20240501_Ehrens-18114-002_GEX3_GEMX \
 --sample=Ehrens-18114-002_GEX3_GEMX \
 --create-bam=false

bsub < count_002.job

Export

scp -r kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/20240501_Ehrens-18114-002_GEX3_GEMX/18114-002/outs/web_summary.html /Users/kxw755/Desktop/Ehrens_StemCells/20240501_Ehrens-18114-002_web_summary.html

scp -r kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/20240501_Ehrens-18114-002_GEX3_GEMX/18114-002/outs/filtered_feature_bc_matrix.h5 /Users/kxw755/Desktop/Ehrens_StemCells/20240501_Ehrens-18114-002_filtered_feature_bc_matrix.h5

scp -r kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/20240501_Ehrens-18114-002_GEX3_GEMX/18114-002/outs/metrics_summary.csv /Users/kxw755/Desktop/Ehrens_StemCells/20240501_Ehrens-18114-002_metrics_summary.csv

Summary

Written on April 24, 2024