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Identification and characterisation of calcitonin receptor isoforms expressed in glioblastoma derived glioma stem and U-87 MG cells.

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Paper

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The authors' code

Shell · 93 lines · 4.7 KB · no license

  2. source discoAnt_params.txt
  4. export PYTHONPATH="$PROGRAMS/cDNA_Cupcake/sequence/:$PYTHONPATH"
  6. echo "Making folders"
  8. mkdir -p $RESULTS
  9. mkdir -p $RESULTS/"$GENE"
  10. mkdir -p $RESULTS/"$GENE"/minimap2
  11. mkdir -p $RESULTS/"$GENE"/bambu
  12. mkdir -p $RESULTS/"$GENE"/bambu_metagene_salmon
  14. ########## ##########
  15. ########## 1. Aligning sample fasta files to reference genome ##########
  16. ########## ##########
  19. echo "minimap2 - Mapping fasta files to genome"
  22. for filename in $FASTA/*.fa
  23. do
  24. base=$(basename $filename .fa)
  25. echo "On sample : $base"
  27. minimap2 -ax splice --splice-flank=yes $REF_HG38/GRCh38.primary_assembly.genome_edit.fa $FASTA/${base}.fa > $RESULTS/"$GENE"/minimap2/${base}.sam
  28. samtools view -S -h -b $RESULTS/"$GENE"/minimap2/${base}.sam | samtools sort - > $RESULTS/"$GENE"/minimap2/${base}_sorted.bam
  29. samtools view -h -F 2308 $RESULTS/"$GENE"/minimap2/${base}_sorted.bam | samtools sort - > $RESULTS/"$GENE"/minimap2/${base}_pri_sorted.bam
  30. done
  32. samtools merge -f $RESULTS/"$GENE"/minimap2/"$GENE"_pri_merged.bam $RESULTS/"$GENE"/minimap2/*_pri_sorted.bam
  33. samtools merge -f $RESULTS/"$GENE"/minimap2/"$GENE"_merged.bam $RESULTS/"$GENE"/minimap2/*_sorted.bam
  35. samtools index $RESULTS/"$GENE"/minimap2/"$GENE"_pri_merged.bam
  36. samtools index $RESULTS/"$GENE"/minimap2/"$GENE"_merged.bam
  38. ########## ##########
  39. ########## 2.a. Correcting and collapsing transcripts with bambu ##########
  40. ########## ##########
  42. Rscript $SCRIPTS/bambu_tx_discovery.R -b $RESULTS/"$GENE"/minimap2/"$GENE"_pri_merged.bam \
  43. -f $REF_HG38/GRCh38.primary_assembly.genome_edit.fa \
  44. -t $REF_HG38/gencode.v41.annotation.gtf \
  45. -o $RESULTS/"$GENE"/bambu
  47. ## Extracting transcripts belonging to the Gene of Interest
  49. cat $RESULTS/"$GENE"/bambu/counts_transcript.txt | grep "$GENE_ID" | awk '{ if ($3 >= 1) print}' > $RESULTS/"$GENE"/bambu/counts_transcript_"$GENE_ID"_count_1.txt
  50. cat $RESULTS/"$GENE"/bambu/counts_transcript_"$GENE_ID"_count_1.txt | awk '{ print $1 }' > $RESULTS/"$GENE"/bambu/counts_transcript_"$GENE_ID"_count_1_transcript.txt
  51. cat $RESULTS/"$GENE"/bambu/extended_annotations.gtf | grep -wf $RESULTS/"$GENE"/bambu/counts_transcript_"$GENE_ID"_count_1_transcript.txt > $RESULTS/"$GENE"/bambu/extended_annotations_"$GENE_ID"_count_1.gtf
  53. ## Editing filtered GTF and counts file for isomix compatibility
  55. sed 's/tx./tx/g' $RESULTS/"$GENE"/bambu/extended_annotations_"$GENE_ID"_count_1.gtf > $RESULTS/"$GENE"/bambu/extended_annotations_"$GENE_ID"_count_1_isomix.gtf
  56. cat $RESULTS/"$GENE"/bambu/counts_transcript_"$GENE_ID"_count_1.txt | sed 's/tx./tx/g' | awk '{ print $1"\t"$3}' > $RESULTS/"$GENE"/bambu/counts_transcript_"$GENE_ID"_count_1_isomix.txt
  59. ########## ##########
  60. ########## 2.b. Creating a transcriptome based on the bambu transcripts ##########
  61. ########## ##########
  63. gffread -w $RESULTS/"$GENE"/bambu/extended_annotations_"$GENE_ID"_count_1.fa -g $REF_HG38/GRCh38.primary_assembly.genome_edit.fa $RESULTS/"$GENE"/bambu/extended_annotations_"$GENE_ID"_count_1.gtf
  64. salmon index -t $RESULTS/"$GENE"/bambu/extended_annotations_"$GENE_ID"_count_1.fa -i $RESULTS/"$GENE"/bambu/extended_annotations_"$GENE_ID"_count_1 -k 31
  66. ########## ##########
  67. ########## 2.c. Re-aligning and quantifying filtered bambu transcripts ##########
  68. ########## ##########
  70. for filename in $FASTA/*.fa
  71. do
  72. base=$(basename $filename .fa)
  73. echo "On sample : $base"
  75. salmon quant -i $RESULTS/"$GENE"/bambu/extended_annotations_"$GENE_ID"_count_1 -l A \
  76. -r $FASTA/${base}.fa -o $RESULTS/"$GENE"/bambu_metagene_salmon/${base}
  78. done
  81. ########## ##########
  82. ########## 3. Annotating transcripts ##########
  83. ########## ##########
  85. gffcompare -r $REF_HG38/gencode.v41.annotation.gtf \
  86. -o $RESULTS/"$GENE"/bambu/extended_annotations_"$GENE_ID"_count_1 $RESULTS/"$GENE"/bambu/extended_annotations_"$GENE_ID"_count_1.gtf
  88. python $PROGRAMS/SQANTI3-4.2/sqanti3_qc.py \
  89. $RESULTS/"$GENE"/bambu/extended_annotations_"$GENE_ID"_count_1.gtf \
  90. $REF_HG38/gencode.v41.annotation.gtf $REF_HG38/GRCh38.primary_assembly.genome_edit.fa \
  91. --cage_peak $REF_HG38/refTSS_v3.3_human_coordinate.hg38.bed \
  92. --polyA_peak $REF_HG38/atlas.clusters.2.0.GRCh38.96.bed --polyA_motif_list $REF_HG38/human.polyA.list.txt \
  93. -d $RESULTS/"$GENE"/sqanti -o $GENE --report skip

discoAnt_v2.sh at commit c8f3c45, no license · at the source

Overview

Authors: Pragya Gupta1, Sebastian GB Furness2, Tahereh Gharbi1, Ric De Paoli‐Iseppi3, Shweta S. Joshi3, Michael Clark3, David L. Hare1, Peter Wookey1
ORCID iDs: Peter Wookey
  1. Department of Medicine (Austin Health, Heidelberg) University of Melbourne Heidelberg Australia
  2. School of Biomedical Sciences, Faculty of Medicine The University of Queensland St Lucia Australia
  3. Department of Anatomy and Physiology University of Melbourne Parkville Australia
Institutions: The University of Melbourne (Australia); Austin Health (Australia); The University of Queensland (Australia)
Journal: FEBS open bio, pages 10.1002/2211-5463.70322
Dates: received 20 April 2026; accepted 31 July 2026; published online 13 August 2026; in print August 2026
Type: Research article · Language: English
License: CC BY
Identifiers: DOI 10.1002/2211-5463.70322 · PMID 42592647 · PMCID PMC13470297 · OpenAlex W7202340914
Open access: gold, a free copy (OpenAlex)
Status: code verified
Categories: genetics / omics (modality), human (organism), other condition (population), cellular / molecular (subfield)
Keywords: alternative splicing, apoptosis, autophagy, calcitonin receptor, CTb Receptor isoform, glioblastoma, nanopore sequencing
Journal subjects: Brain Cancer, Alternative Splicing, Cancer and Oncology
Topic: Neuropeptides and Animal Physiology (Cellular and Molecular Neuroscience, Neuroscience), according to OpenAlex
Funding: Australian Research Council (FT180100543); University of Melbourne
Citations: not cited yet (Europe PMC); 91 references in the paper
Research resources: Australia RRID:CVCL_0022, JK2 RRID:CVCL_VS46, PB1 RRID:CVCL_VS49, SB2b RRID:CVCL_VS52, HGG cell lines [WK1 RRID:CVCL_VS54

Abstract

Glioblastoma (GBM) is a highly lethal brain cancer in which the calcitonin receptor (CT Receptor), encoded by the CALCR gene, is expressed in 78–88% of patient biopsies. Here, we investigate whether the CT Receptor plays a role in cancer cell survival. In cancer cell lines, knockdown of CALCR disrupts the cell cycle and induces apoptosis, supporting an essential pro‐survival role. The CALCR gene produces three main transcripts in humans, of which Transcript 1 encodes CALCR b mRNA including exon 10 and is translated into the CTb Receptor isoform, and Transcripts 2 and 3 which are translated into the CTa Receptor. CALCR b expression is conserved across a diverse range of mammalian species. We examined the expression of all CT Receptor isoforms (CALCR total) and CALCR b expression in four high‐grade glioma stem‐like cell lines and in U‐87 MG glioblastoma cells. Using qPCR, we observed stable levels of both CALCR total and CALCR b expression under conditions of autophagy or apoptosis, consistent with a requirement for CALCR b in cell survival. As alternative splicing (AS) of key genes in cancers confers tumour resilience, we investigated AS of CALCR transcript 2 using long‐read nanopore sequencing. Unexpectedly, we discovered a novel AS event causing inclusion of exon 10 within Transcript 2 in all glioblastoma cell lines investigated. This finding, together with stable CALCR b expression under cellular stress and the finding by other groups confirming that knockdown of CT Receptor compromises cell survival, implicates the CTb Receptor as a potential oncoprotein.

Reproduced under the paper's license (CC BY), from the paper cited above.

Repository

Its files are read in the Code ↔ Paper reader above.

shwetajoshi-15/discoAnt

License: none: the authors keep all their rights
State: the link answers, verified on 27 September 2026
Evidence: files inventoried
Commit: c8f3c4572e219070169f676faeb9302823ccce9c, 19 September 2023
Languages: Shell (4), R (1)
Size: 21 files, 5 scripts
Software Heritage: not archived
Found in: the text, “Bioinformatic analysis”
Holds: README
Not found: license file, CITATION.cff, environment file, tests, continuous integration, documentation
Tools: Salmon (3 files), SAMtools (3 files), BEDTools (1 file), tidyverse (1 file)
Availability: 1 check, the latest on 27 September 2026: the link answers
  • 27 September 2026: the link answers
6 files
At the source: github.com/shwetajoshi-15/discoAnt

Tracing map

Proposed by the machine: these links were found in the paper and verified at the source, without human review. The map will receive a Zenodo DOI once one of the paper's authors has validated it with their ORCID.

What the map holds:

  • 1 repository of the authors' code, each at its verified commit, with its license and how the link was found in the paper;
  • 5 scripts, each with its path and the digest of its content;
  • no match between paragraphs and code yet;
  • neither the text of the paper nor the code itself.

Its JSON (tracing-map.json) is deposited on Zenodo with its DOI once the map is validated.

Data

Datasets cited

Data availability statement

The data that support the findings of this study are openly available in Figshare (https://figshare.com/projects/Dataset_Manuscript_to_FEBSOPEN/279239 (https://url.au.m.mimecastprotect.com/s/2oFKCyoNK5U6MJNw1cZfwfxp2aB?domain=figshare.com)).

Reproduced under the paper's license (CC BY), from the paper cited above.

Versions

The history of this record: each version stored by the harvester or made by a correction of its authors or of the maintainers of its code, and what changed in its facts. The texts of the paper (its abstract, its availability statements) are not part of it; versions that changed only those are not listed.

Version 1, 27 September 2026: the first record

Recorded: type, language, journal, pages, dates, 8 authors, 7 keywords, 2 funders, 89 references, 5 RRIDs.

Cite

This paper

Gupta, P., Furness, S. G., Gharbi, T., De Paoli‐Iseppi, R., Joshi, S. S., Clark, M., Hare, D. L., & Wookey, P. (2026). Identification and characterisation of calcitonin receptor isoforms expressed in glioblastoma derived glioma stem and U-87 MG cells. FEBS open bio, 10.1002/2211-5463.70322. https://doi.org/10.1002/2211-5463.70322

BibTeX

@article{gupta2026identification,
author = {Gupta, Pragya and Furness, Sebastian GB and Gharbi, Tahereh and De Paoli‐Iseppi, Ric and Joshi, Shweta S. and Clark, Michael and Hare, David L. and Wookey, Peter},
title = {{Identification and characterisation of calcitonin receptor isoforms expressed in glioblastoma derived glioma stem and U-87 MG cells}},
journal = {FEBS open bio},
year = {2026},
month = aug,
pages = {10.1002/2211--5463.70322},
publisher = {Wiley},
issn = {2211-5463},
doi = {10.1002/2211-5463.70322},
url = {https://doi.org/10.1002/2211-5463.70322},
pmid = {42592647},
pmcid = {PMC13470297}
}

RIS

TY - JOUR
AU - Gupta, Pragya
AU - Furness, Sebastian GB
AU - Gharbi, Tahereh
AU - De Paoli‐Iseppi, Ric
AU - Joshi, Shweta S.
AU - Clark, Michael
AU - Hare, David L.
AU - Wookey, Peter
TI - Identification and characterisation of calcitonin receptor isoforms expressed in glioblastoma derived glioma stem and U-87 MG cells
T2 - FEBS open bio
J2 - FEBS Open Bio
PY - 2026
DA - 2026/08/13
SP - 10.1002/2211
EP - 5463.70322
SN - 2211-5463
PB - Wiley
DO - 10.1002/2211-5463.70322
UR - https://doi.org/10.1002/2211-5463.70322
LA - en
ER -

CSL-JSON

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"title": "Identification and characterisation of calcitonin receptor isoforms expressed in glioblastoma derived glioma stem and U-87 MG cells",
"container-title": "FEBS open bio",
"author": [
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"given": "Pragya"
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],
"container-title-short": "FEBS Open Bio",
"page": "10.1002/2211-5463.70322",
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"PMID": "42592647",
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"ISSN": "2211-5463",
"publisher": "Wiley",
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"date-parts": [
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13
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