Gfas_Syms_CellRanger_Analysis

Goal: To re-analyze the 10X single cell data with the combined genomic references of Galaxea fasicularis and potential symbionts

Reference Genomes:

Galaxea fasicularis v1
- From Reef Genomics
- Download site
Durisdinum trenchii (SCF082)
- Dougan et al. 2022
- Download site
Breviolum (Symbiodinium minutum)
- Download site
Cladocopium
- Download site
- alternate Download site
  - Reference: Liu H, Stephens TG, González-Pech RA, Beltran VH, Lapeyre B, Bongaerts P, Cooke I, Aranda M, Bourne DG, Forêt S, Miller DJ, van Oppen MJH, Voolstra CR, Ragan MA, Chan CX Symbiodinium genomes reveal adaptive evolution of functions related to coral-dinoflagellate symbiosis. Commun Biol. 2018;1():95. doi: 10.1038/s42003-018-0098-3
Symbiodinium microadriaticum
- Download site
- alternate Download Site
  - Reference: Aranda M, Li Y, Liew YJ, Baumgarten S, Simakov O, Wilson MC, Piel J, Ashoor H, Bougouffa S, Bajic VB, Ryu T, Ravasi T, Bayer T, Micklem G, Kim H, Bhak J, LaJeunesse TC, Voolstra CR Genomes of coral dinoflagellate symbionts highlight evolutionary adaptations conducive to a symbiotic lifestyle. Sci Rep. 2016 Dec 22;6():39734. doi: 10.1038/srep39734

File upload and organization

mkdir sym_genomes
cd sym_genomes/

scp -r symA3_symb.gff.gz kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/sym_genomes/symA3_symb.gff.gz
scp -r symC_40.gff.gz kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/sym_genomes/symC_40.gff.gz
scp -r symbB.v1.2.augustus.gff3.gz kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/sym_genomes/symbB.v1.2.augustus.gff3.gz

scp -r symA3_37.fasta.gz kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/sym_genomes/symA3_37.fasta.gz
scp -r symbB.v1.0.genome.fa.gz kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/sym_genomes/symbB.v1.0.genome.fa.gz
scp -r symC_scaffold_40.fasta.gz kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/sym_genomes/symC_scaffold_40.fasta.gz

the sym C and A gff files did not have exon reads and cell ranger could not make the reference genome. I will download the following to see if these work

scp -r Clago1_AssemblyScaffolds_Repeatmasked.fasta.gz kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/sym_genomes/Clago1_AssemblyScaffolds_Repeatmasked.fasta.gz
scp -r Clago1_GeneCatalog_genes_20200812.gff3.gz kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/sym_genomes/Clago1_GeneCatalog_genes_20200812.gff3.gz

scp -r Symmic1_AssemblyScaffolds_Repeatmasked.fasta.gz kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/sym_genomes/Symmic1_AssemblyScaffolds_Repeatmasked.fasta.gz
scp -r Symmic1_all_genes_20180603.gff3.tgz kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/sym_genomes/Symmic1_all_genes_20180603.gff3.tgz

Unzip and convert to gff3

gunzip symA3_symb.gff.gz
mv symA3_symb.gff symA3_symb.gff3

gunzip symbB.v1.2.augustus.gff3.gz

gunzip symC_40.gff.gz
mv symC_40.gff symC_40.gff3

gunzip Dtrenchii_SCF082_ANNOT_gff.gz
mv Dtrenchii_SCF082_ANNOT_gff Dtrenchii_SCF082_ANNOT.gff3

gunzip symA3_37.fasta.gz
gunzip symbB.v1.0.genome.fa.gz
gunzip symC_scaffold_40.fasta.gz
gunzip Dtrenchii_SCF082_ASSEMBLY_fasta.gz

mv Dtrenchii_SCF082_ASSEMBLY_fasta Dtrenchii_SCF082_ASSEMBLY.fasta

gunzip Clago1_AssemblyScaffolds_Repeatmasked.fasta.gz
gunzip Clago1_GeneCatalog_genes_20200812.gff3.gz

tar -xvzf Symmic1_all_genes_20180603.gff3.tgz
cd /nethome/kxw755/sym_genomes/global/projectb/sandbox/fungal/data/Symbiodinium_microadriaticum/Symmic1/download
cp Symmic1.ExternalModels.gff3 ../../../../../../../../

gunzip Symmic1_AssemblyScaffolds_Repeatmasked.fasta.gz

these gff3 files seem to have all the genomic information rather than just CDS

Converting this GFF3 file to GTF:

nano convert.job

#!/bin/bash

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

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

module load cufflinks/2.2.1 

gffread Dtrenchii_SCF082_ANNOT.gff3 -T -o Dtrenchii_SCF082_ANNOT.gtf

bsub < convert.job

nano convert2.job

#!/bin/bash

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

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

module load cufflinks/2.2.1 

gffread symA3_symb.gff3 -T -o symA3_symb.gtf
gffread symbB.v1.2.augustus.gff3 -T -o symbB.v1.2.augustus.gtf
gffread symC_40.gff3 -T -o symC_40.gtf

bsub < convert2.job

nano convert3.job

#!/bin/bash

#BSUB -J scSeq_convertGTF
#BSUB -q general
#BSUB -P dark_genes
#BSUB -n 6
#BSUB -W 12:00
#BSUB -u kxw755@earth.miami.edu
#BSUB -o syms_convertGTF3.out
#BSUB -e syms_convertGTF3.err

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

module load cufflinks/2.2.1 

gffread Clago1_GeneCatalog_genes_20200812.gff3 -T -o Clago1_GeneCatalog_genes_20200812.gtf
gffread Symmic1.ExternalModels.gff3 -T -o Symmic1.ExternalModels.gtf

bsub < convert3.job

Making combined genome reference

nano mkgtf_syms.job

#!/bin/bash

#BSUB -J scSeq_mkgtf_syms
#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 gfas_syms_mkgtf_v2.out
#BSUB -e gfas_syms_mkgtf_v2.err

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

cellranger mkref \
--genome=gfas_1.0 --fasta=/nethome/kxw755/Gfas_v1/gfas_final_1.0.fasta --genes=/nethome/kxw755/Gfas_v1/gfas_1.0.filtered.gtf \
--genome=symA --fasta=/nethome/kxw755/sym_genomes/Symmic1_AssemblyScaffolds_Repeatmasked.fasta --genes=/nethome/kxw755/sym_genomes/Symmic1.ExternalModels.gtf \
--genome=symB --fasta=/nethome/kxw755/sym_genomes/symbB.v1.0.genome.fa --genes=/nethome/kxw755/sym_genomes/symbB.v1.2.augustus.gtf \
--genome=symC --fasta=/nethome/kxw755/sym_genomes/Clago1_AssemblyScaffolds_Repeatmasked.fasta --genes=/nethome/kxw755/sym_genomes/Clago1_GeneCatalog_genes_20200812.gtf \
--genome=symD --fasta=/nethome/kxw755/sym_genomes/Dtrenchii_SCF082_ASSEMBLY.fasta --genes=/nethome/kxw755/sym_genomes/Dtrenchii_SCF082_ANNOT.gtf \
--memgb 20

bsub < mkgtf_syms.job

error:

Fatal LIMIT error: the number of junctions to be inserted on the fly =2833619 is larger than the limitSjdbInsertNsj=1000000
Fatal LIMIT error: the number of junctions to be inserted on the fly =2833619 is larger than the limitSjdbInsertNsj=1000000
SOLUTION: re-run with at least --limitSjdbInsertNsj 2833619

Mar 26 16:12:54 ...... FATAL ERROR, exiting

Maybe lets try this without symA and see if this frees up the memory/RAM usage

nano mkgtf_syms2.job

#!/bin/bash

#BSUB -J scSeq_mkgtf_syms2
#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 gfas_syms_mkgtf_v2.out
#BSUB -e gfas_syms_mkgtf_v2.err

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

cellranger mkref \
--genome=gfas_1.0 --fasta=/nethome/kxw755/Gfas_v1/gfas_final_1.0.fasta --genes=/nethome/kxw755/Gfas_v1/gfas_1.0.filtered.gtf \
--genome=symB --fasta=/nethome/kxw755/sym_genomes/symbB.v1.0.genome.fa --genes=/nethome/kxw755/sym_genomes/symbB.v1.2.augustus.gtf \
--genome=symC --fasta=/nethome/kxw755/sym_genomes/Clago1_AssemblyScaffolds_Repeatmasked.fasta --genes=/nethome/kxw755/sym_genomes/Clago1_GeneCatalog_genes_20200812.gtf \
--genome=symD --fasta=/nethome/kxw755/sym_genomes/Dtrenchii_SCF082_ASSEMBLY.fasta --genes=/nethome/kxw755/sym_genomes/Dtrenchii_SCF082_ANNOT.gtf 

bsub < mkgtf_syms2.job

Fatal LIMIT error: the number of junctions to be inserted on the fly =1992803 is larger than the limitSjdbInsertNsj=1000000
Fatal LIMIT error: the number of junctions to be inserted on the fly =1992803 is larger than the limitSjdbInsertNsj=1000000
SOLUTION: re-run with at least --limitSjdbInsertNsj 1992803

Mar 27 11:04:18 ...... FATAL ERROR, exiting

okay - according to this link I need to adjust a parameter in the STAR file, but I don’t think I have access. I am going to just map the symbionts with out the Galaxea genome and see what hits.

nano mkgtf_syms_only.job

#!/bin/bash

#BSUB -J scSeq_mkgtf_symsonly
#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 syms_mkgtf_only.out
#BSUB -e syms_mkgtf_only.err

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

cellranger mkref \
--genome=symA --fasta=/nethome/kxw755/sym_genomes/Symmic1_AssemblyScaffolds_Repeatmasked.fasta --genes=/nethome/kxw755/sym_genomes/Symmic1.ExternalModels.gtf \
--genome=symB --fasta=/nethome/kxw755/sym_genomes/symbB.v1.0.genome.fa --genes=/nethome/kxw755/sym_genomes/symbB.v1.2.augustus.gtf \
--genome=symC --fasta=/nethome/kxw755/sym_genomes/Clago1_AssemblyScaffolds_Repeatmasked.fasta --genes=/nethome/kxw755/sym_genomes/Clago1_GeneCatalog_genes_20200812.gtf \
--genome=symD --fasta=/nethome/kxw755/sym_genomes/Dtrenchii_SCF082_ASSEMBLY.fasta --genes=/nethome/kxw755/sym_genomes/Dtrenchii_SCF082_ANNOT.gtf 

bsub < mkgtf_syms_only.job

Rerunning Count with the combined genome

Run 1

cd /nethome/kxw755/20231004_SingleCell_DG/

nano count_W045_deep_syms.job

#BSUB -J count_W045_deep_syms
#BSUB -q general
#BSUB -P dark_genes
#BSUB -n 6
#BSUB -W 120:00
#BSUB -u kxw755@earth.miami.edu
#BSUB -o count.out
#BSUB -e count.err
#BSUB -B
#BSUB -N
###################################################################

cellranger count \
 --id=W-045_1_deep_syms \
 --transcriptome=/nethome/kxw755/sym_genomesgfas_1.0_and_symA3_and_symB_and_symC_and_symD \
 --fastqs=/nethome/kxw755/20231004_SingleCell_DG \
 --sample=AndradeRodriguez-15275-001_GEX3

bsub < count_W045_deep_syms.job

Export:

scp -r kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/20231004_SingleCell_DG/W-045_1_deep_combgenome/outs/filtered_feature_bc_matrix.h5 /Users/kevinwong/MyProjects/DarkGenes_Bleaching_Comparison/output/CellRanger/20240326_W045_combgeno_filt_feature_bc_matrix.h5

scp -r kxw755@pegasus.ccs.miami.edu:/nethome/kxw755/20231004_SingleCell_DG/W-045_1_deep_combgenome/outs/web_summary.html /Users/kevinwong/MyProjects/DarkGenes_Bleaching_Comparison/output/CellRanger/20240326_W045_combgeno_web_summary.html

Written on January 31, 2024