RNA-seq on sage

TODO on sage

check the alignment of the reads to the annotation which sent from Munich is very bad, using the reference X14112 instead, find the CMV-GFP in the genome. Using alignment to detect the overall alignment rate to X14112 and chrHsv1_s17.

commands on sage

#under sage
ln -s /home/jhuang/Tools/nf-core-rnaseq-3.12.0/ rnaseq
[jhuang@sage Data_Caroline_RNAseq_wt_timecourse] nextflow run rnaseq/main.nf --input samplesheet_wt_timecourse.csv --outdir results_GRCh38 --genome GRCh38 -profile test_full  -resume   --max_memory 256.GB --max_time 2400.h        --aligner 'star_salmon' --skip_multiqc
[jhuang@sage Data_Caroline_RNAseq_wt_timecourse] nextflow run rnaseq/main.nf --input samplesheet_wt_timecourse.csv --outdir results_chrHsv1  --fasta chrHsv1_s17.fasta --gtf chrHsv1_s17.gtf  -profile test_full -resume  --max_memory 256.GB --max_time 2400.h     --save_reference    --aligner 'star_salmon'    --gtf_extra_attributes 'gene_id' --gtf_group_features 'transcript_id' --featurecounts_group_type 'gene_id' --featurecounts_feature_type 'transcript'  --skip_rseqc --skip_dupradar --skip_preseq --skip_biotype_qc --skip_deseq2_qc --skip_multiqc
[jhuang@sage Data_Caroline_RNAseq_brain_organoids] nextflow run rnaseq/main.nf --input samplesheet_brain_organoids.12.csv --outdir results_GRCh38 --genome GRCh38 -profile test_full  -resume   --max_memory 256.GB --max_time 2400.h        --aligner 'star_salmon' --skip_multiqc
[jhuang@sage Data_Caroline_RNAseq_brain_organoids] nextflow run rnaseq/main.nf --input samplesheet_brain_organoids.12.csv --outdir results_chrHsv1  --fasta chrHsv1_s17.fasta --gtf chrHsv1_s17.gtf  -profile test_full -resume  --max_memory 256.GB --max_time 2400.h     --save_reference    --aligner 'star_salmon'    --gtf_extra_attributes 'gene_id' --gtf_group_features 'transcript_id' --featurecounts_group_type 'gene_id' --featurecounts_feature_type 'transcript'  --skip_rseqc --skip_dupradar --skip_preseq --skip_biotype_qc --skip_deseq2_qc --skip_multiqc
#Processing *.umi_extract.fastq.gz
(rnaseq) [jhuang@sage Data_Manja_RNAseq_Organoids_Virus]$ nextflow run rnaseq/main.nf --input samplesheet.umi_extract.csv --outdir results_chrHsv1  --fasta chrHsv1_s17.fasta --gtf chrHsv1_s17.gtf  -profile test_full -resume  --max_memory 256.GB --max_time 2400.h     --save_reference    --aligner 'star_salmon'    --gtf_extra_attributes 'gene_id' --gtf_group_features 'transcript_id' --featurecounts_group_type 'gene_id' --featurecounts_feature_type 'transcript'  --skip_rseqc --skip_dupradar --skip_preseq --skip_biotype_qc --skip_deseq2_qc --skip_multiqc
#Processing raw data prepared with umi protocol
(rnaseq) [jhuang@sage Data_Manja_RNAseq_Organoids_Virus]$ nextflow run rnaseq/main.nf --input samplesheet.csv --outdir results_chrHsv1  --fasta chrHsv1_s17.fasta --gtf chrHsv1_s17.gtf  --with_umi --umitools_extract_method "regex" --umitools_bc_pattern "^(?P<umi_1>.{12}).*" --umitools_dedup_stats    -profile test_full -resume  --max_memory 256.GB --max_time 2400.h     --save_reference    --aligner 'star_salmon'    --gtf_extra_attributes 'gene_id' --gtf_group_features 'transcript_id' --featurecounts_group_type 'gene_id' --featurecounts_feature_type 'transcript'  --skip_rseqc --skip_dupradar --skip_preseq --skip_biotype_qc --skip_deseq2_qc --skip_multiqc --min_mapped_reads 0
#Debug the following error: added "--minAssignedFrags 0 \\" to modules/nf-core/salmon/quant/main.nf option "salmon quant" and added "--min_mapped_reads 0" in the nextflow command above
#hits: 0; hits per frag:  0[2023-10-20 11:35:22.944] [jointLog] [warning] salmon was only able to assign 0 fragments to transcripts in the index, but the minimum number of required assigned fragments (--minAssignedFrags) was 1. This could be indicative of a mismatch between the reference and sample, or a very bad sample.  You can change the --minAssignedFrags parameter to force salmon to quantify with fewer assigned fragments (must have at least 1).
(rnaseq) [jhuang@sage Data_Denise_LT_RNAseq]$ nextflow run rnaseq/main.nf --input samplesheet.csv --outdir results_GRCh38 --genome GRCh38 -profile test_full  -resume   --max_memory 256.GB --max_time 2400.h    --save_align_intermeds --save_unaligned    --aligner 'star_salmon' --skip_multiqc
(rnaseq) [jhuang@sage Data_Samira_RNAseq]$ nextflow run rnaseq/main.nf --input samplesheet.csv --outdir results_GRCh38 --genome GRCh38 -profile test_full  -resume   --max_memory 256.GB --max_time 2400.h    --save_align_intermeds --save_unaligned    --aligner 'star_salmon'
(rnaseq) [jhuang@sage Data_Manja_RNAseq_Organoids]$ nextflow run rnaseq/main.nf --input samplesheet.csv --outdir results_GRCh38 --genome GRCh38   --with_umi --umitools_extract_method "regex" --umitools_bc_pattern "^(?P<umi_1>.{12}).*"         -profile test_full  -resume   --max_memory 256.GB --max_time 2400.h    --save_align_intermeds --save_unaligned --save_reference    --aligner 'star_salmon' --pseudo_aligner 'salmon'
(rnaseq) [jhuang@sage Data_Manja_RNAseq_Organoids_Virus]$ nextflow run rnaseq/main.nf --input samplesheet.csv --outdir results_chrHsv1_s17    --fasta "/home/jhuang/DATA/Data_Manja_RNAseq_Organoids_Virus/chrHsv1_s17.fasta" --gtf "/home/jhuang/DATA/Data_Manja_RNAseq_Organoids_Virus/chrHsv1_s17.gtf"   --with_umi --umitools_extract_method "regex" --umitools_bc_pattern "^(?P<umi_1>.{12}).*" --umitools_dedup_stats    --skip_rseqc --skip_dupradar --skip_preseq    -profile test_full -resume  --max_memory 256.GB --max_time 2400.h    --save_align_intermeds --save_unaligned --save_reference    --aligner 'star_salmon'    --gtf_extra_attributes 'gene_id' --gtf_group_features 'transcript_id' --featurecounts_group_type 'gene_id' --featurecounts_feature_type 'transcript' --skip_multiqc
ln -s ~/Tools/rnaseq/assets/multiqc_config.yaml multiqc_config.yaml
multiqc -f --config multiqc_config.yaml . 2>&1
rm multiqc_config.yaml

reference on sage

/home/jhuang/REFs/Homo_sapiens/Ensembl/GRCh38
/home/jhuang/REFs/Homo_sapiens/hg38-blacklist.bed
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