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| Status |
Public on Nov 16, 2024 |
| Title |
Medulloblastoma frozen tumor, Non-WNT/Non-SHH, M087RB11 |
| Sample type |
SRA |
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| Source name |
Brain Tumor
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Brain Tumor
cell type: Medulloblastoma
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA were extracted from frozen MB samples using the standard phenol-chloroform procedure. The quality and quantity of DNA were assessed using Nanodrop (Thermo Fisher Scientific) and Qubit (Thermo Fisher Scientific). Quantification of DNA was performed using the Qubit Fluorometer (Quant-iT dsDNA BR Assay Kit; Thermo Fisher Scientific) and quality assessment was carried out using TapeStation (Genomic DNA screenTape; Agilent) following the manufacturer’s instructions.
For whole-genome Nanopore sequencing, DNA libraries were prepared using the SQK-RBK004 rapid barcoding kit from Oxford Nanopore Technologies. Two types of flow cells were used: MinION R9.4.1 (FLO-MIN106D) and Flongle R9.4.1 (FLO-FLG001). For MinION flow cells, 400 ng of DNA were tagmented, barcoded, repaired, and pooled in batches of six samples before adaptor ligation. Sequencing involved multiplexing six libraries, with each batch taking 48 to 72 hours per MinION R9.4.1 flow cell. For Flongle flow cells, the same SQK-RBK004 kit (Oxford Nanopore Technologies) was used, but 200 ng of DNA per tumor sample was sequenced individually on a Flongle flow cell for 24 hours. All sequencing was performed on MinION Mk1B or Mk1C devices, and sequencing runs were initiated and monitored using MinKnow software from Oxford Nanopore Technologies.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
MinION |
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| Data processing |
Downstream analysis relied on a custom bioinformatic pipeline called NanoCliD, available on GitHub (https://github.com/InstituteCurieClinicalBioinformatics/NanoCliD). Methylation calling was performed using Nanopolish 0.14.0, and the Guppy 5.0.17 data processing toolkit facilitated the conversion of FAST5 files to FASTQ files. Sequences were aligned to the reference genome hg19 with Minimap 2.24-r1122. CNV Calling was performed, adapted from NanoDX7, using QDNASeq and DeepTools v3.5.1 (Fig 1b). The obtained copy number profiles were then manually analyzed, focusing on key alterations encountered in medulloblastoma which are chromosome 6 monosomy, GLI2, MYC, MYCN, MYCL, SNCAIP and OTX2 amplifications, loss of chromosome 9q and 10q and isochromosome 17.
For each sample, Nanopore sequencing methylation data werre compared to the 3,905 publicly available Illumina arrays (Accession series GSE109381 updated version; https://www.ncbi.nlm.nih.gov/gds), corresponding to the v11b4 Heidelberg classifier (https://www.molecularneuropathology.org/mnp/). The NanoDx pipeline7 was integrated into NanoCliD for tumor classification. The CpG called during Nanopore sequencing were cross-referenced with those from the 850,000 covered by the EPIC array to retain only common sites. Random forest algorithm was employed to obtain methylation profiling, enabling tumor assignment to a group based on the analyzed CpG overlapping those of Illumina arrays (as illustrated in Fig. 2). The score, representing the sample's passage through the algorithm's decision trees, was obtained by counting each class returned by each tree. As described by other teams before, Aa random forrest score above 0.15 was considered satisfactory for reliable classification7. Uniform Manifold Approximation and Projection (UMAP) (Fig. 2) and t-distributed Stochastic Neighbor Embedding (t-SNE) were also generated for each sample for visualization.
Assembly: hg19
Supplementary files format and content: MethylationTables.tar.gz : list of tsv files obtained after methylation calling
Library strategy: Nanopore whole genome long read sequencing
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| Submission date |
Oct 22, 2024 |
| Last update date |
Nov 16, 2024 |
| Contact name |
Mathilde Filser |
| E-mail(s) |
[email protected]
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| Organization name |
Institut Curie
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| Department |
Genetics Department
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| Street address |
26 rue d'Ulm
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| City |
Paris |
| ZIP/Postal code |
75005 |
| Country |
France |
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| Platform ID |
GPL24106 |
| Series (1) |
| GSE280038 |
Nanopore Sequencing as a Cutting-Edge Technology for Medulloblastoma Classification |
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| Relations |
| BioSample |
SAMN44378584 |
| SRA |
SRX26455547 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8586189_M087RB11.methylation_calls_frequency.tsv.gz |
36.6 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
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