📄 Methods & data

How the results on this site were produced, and where the underlying samples came from.

RNA-seq pipeline

Tumor RNA-seq was processed with the nf-core/oncoanalyser pipeline (Hartwig tooling) in an RNA-only, tumor-only configuration — there is no matched normal and no DNA sequencing in this analysis, so all DNA/germline modules are intentionally disabled.

FASTQ → fastp (trim) → STAR (align) → Isofox (expression + fusions) → CUPPA (tissue of origin)

Isofox produces per-gene and per-transcript expression, gene fusions, and splicing metrics.
CUPPA predicts molecular tissue of origin from the RNA features.
Quality control uses FastQC and alignment metrics aggregated with MultiQC.

GTEx comparison: the same two RNA samples were also reprocessed through the GTEx V11 pipeline (STAR → RSEM → RNA-SeQC) so their expression can be compared directly against GTEx normal tissues and PCAWG tumors — see Expression vs GTEx.

Immune repertoire (TCR/BCR)

TRUST4 (Song et al., Nat Methods 2021) was run on the three RNA BAMs to de-novo assemble T-cell and B-cell receptor CDR3 sequences, as an independent cross-check of oncoanalyser's CIDER module (both reconstruct the IG/TR repertoire from the same reads). Candidate reads over the V/D/J/C loci plus unmapped mates are extracted (with --abnormalUnmapFlag, required for these BAMs), assembled, and annotated against IMGT. Clone counts are normalized to library size (per million Isofox fragments) so samples of different depth compare fairly — see Immune repertoire.

Interpretation & limits: bulk RNA-seq samples the repertoire shallowly and is biased toward highly-expressed (plasma) cells, so the low clone overlap between the metastasis and normal lung is largely sampling rather than biology, and the "met-enriched" candidate clones are hypothesis-generating leads — sequence alone cannot establish that an antibody recognizes the tumor.

Sequencing quality control

Per-sample RNA-seq quality metrics from Isofox; both tumor samples pass QC.

Sample	QC	Total fragments	Duplicates	Spliced genes	Read length	Median fragment
Left hand (primary)	PASS	41,404,403	23.5%	21,354	76 bp	299 bp
Lung metastasis	PASS	124,808,404	50.5%	26,729	101 bp	133 bp
Normal lung (baseline)	PASS	111,295,463	55.6%	25,638	101 bp	127 bp

On the numbers: the two Treehouse libraries (lung metastasis and normal lung) have higher duplicate rates and shorter fragments than the Tempus primary, consistent with their low-input (100 ng) preparation. All three samples pass and yield tens of thousands of expressed genes. The normal lung sample is an in-patient baseline processed identically to the tumors — a specificity control (for example, the diagnostic EWSR1::KLF15 fusion is present in the tumor but absent in normal lung), not itself a tumor.

Open full MultiQC report ↗

Samples shown on this site

3 RNA-seq samples were reprocessed locally and are shown here — two of the catalogued tumor specimens plus a normal-lung baseline (a within-patient control, not itself a catalogued tumor):

Left hand (primary) (SUH-21-014093, FFPE) — driver fusion EWSR1::KLF15.
Lung, left lower lobe (metastasis) (SP-22-081918-C, Originally Frozen) — driver fusion EWSR1::KLF15.
Normal lung (baseline) — non-tumor expression baseline and fusion specificity control (from the same Treehouse delivery as the metastasis).

Full sample catalogue

#	Accession	Site	Phase	Type	Diagnosis	Fusion	NGS	Vendor
1	SUH-21-014093	Left hand	Primary / earliest known specimen	FFPE	MEC	EWSR1::KLF15	WES, RNA-seq, single cell	Tempus; NeoGenomics; BostonGene
2	LPS-22-04880	Lung, right lower lobe	Pulmonary metastatic / recurrent lesion	FFPE	MEC	Not yet documented	N/A	N/A
3	CF-01500-2	Lung, left lower lobe	Pulmonary metastatic / recurrent lesion	Cryopreserved	MEC	EWSR1::KLF15	WES, RNA-seq	BGI Genomics
4	SP-22-081918-C	Lung, left lower lobe	Pulmonary metastatic / recurrent lesion	Originally Frozen	MEC	EWSR1::KLF15	RNA-seq	Covance by Labcorp
5	SP-22-081918-A	Lung, right lower lobe	Pulmonary metastatic / recurrent lesion	FFPE	MEC	Not yet documented	WES, RNA-seq, single cell	Tempus; BostonGene
6	SP-22-081918-B	Lung, right upper lobe	Pulmonary metastatic / recurrent lesion	FFPE	MEC	Not yet documented	N/A	N/A

Diagnosis abbreviations are as recorded in the clinical sample sheet. Several specimens have whole-exome sequencing (WES) held at external vendors; those data are not part of this analysis.

Contact Us

Please contact us with any questions or comments.