{ "version": "https:\/\/jsonfeed.org\/version\/1.1", "title": "Konstantin Chernyshov: posts tagged 2023", "_rss_description": "Konstantin Chernyshov, manager, bioinformatic, leader in biotech", "_rss_language": "en", "_itunes_email": "", "_itunes_categories_xml": "", "_itunes_image": "https:\/\/kostachernyshov.com\/blog\/pictures\/userpic\/userpic-square@2x.jpg?1731242071", "_itunes_explicit": "no", "home_page_url": "https:\/\/kostachernyshov.com\/blog\/tags\/2023\/", "feed_url": "https:\/\/kostachernyshov.com\/blog\/tags\/2023\/json\/", "icon": "https:\/\/kostachernyshov.com\/blog\/pictures\/userpic\/userpic@2x.jpg?1731242071", "authors": [ { "name": "Konstantin Chernyshov", "url": "https:\/\/kostachernyshov.com\/blog\/", "avatar": "https:\/\/kostachernyshov.com\/blog\/pictures\/userpic\/userpic@2x.jpg?1731242071" } ], "items": [ { "id": "38", "url": "https:\/\/kostachernyshov.com\/blog\/all\/geomx-and-mycosis-fungoides\/", "title": "GeoMx and mycosis fungoides", "content_html": "

Project on lymphoma<\/h2>\n

Mycosis fungoides, or cutaneous T-cell lymphoma, is a cancer in which deranged T lymphocytes start proliferating in the skin. The client carried out the wet part of the study using GeoMx technology and ordered the analysis from us at BostonGene. I led the project, worked out the annotations and the nuances of the technology and communicated with the client, while Lesha did the quality control and analysis.<\/p>\n

GeoMx technology and files<\/h2>\n

Original article: Multiplex digital spatial profiling of proteins and RNA in fixed tissue, 2020<\/a><\/p>\n

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\n\"\"\n\"\"\n<\/div>\n
The essence of GeoMx technology and an image of a piece of skin from the study and the ROIs (regions of interest) on this piece.<\/div>\n<\/div>\n

The essence of GeoMx technology:<\/p>\n

    \n
  1. There is a pre-prepared set of RNA probes for thousands of genes. These probes are connected by a UV linker to a barcode oligonucleotide. One gene we care about = one barcode.<\/li>\n
  2. The biopsy is prepared, placed on a slide, treated with RNA probes, and hybridization takes place.<\/li>\n
  3. Then the slide is placed into a machine with a camera, and on the microphoto shown on the machine’s screen, the researcher marks the region of interest.<\/li>\n
  4. Then the machine blasts this outlined region with UV light, the UV linker falls apart, and the machine sucks up the barcodes.<\/li>\n
  5. The barcodes are sequenced, even on a regular sequencer, and reads are produced.<\/li>\n
  6. The reads are matched against a pre-prepared barcode–gene panel. The number of reads from each barcode is basically transformed into the signal intensity of a particular RNA.<\/li>\n<\/ol>\n

    Therefore when we received raw sequencing files it became clear that this was not what we needed. We were sent oligo‑DNA barcodes that by themselves carry no information about genes and need to be matched to pairs of barcode‑gene via a special pipeline. The output files are Digital Count Conversion (DCC).<\/p>\n

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    \n\"\"\n\"\"\n<\/div>\n
    How DCC files are obtained from the GeoMx manual and the Illumina pipeline.<\/div>\n<\/div>\n

    You can process fastq files yourself via Illumina’s BaseSpace GeoMx NGS Pipeline is distributed as a pipeline on BaseSpace<\/a>. It seems this doesn’t require special access, but it costs money and is not trivial at all. So I decided to write to the collaborator and ask if they already had DCC files. The DCC files were received the next day. Sometimes it’s easiest to simply ask.<\/p>\n

    Annotation<\/h2>\n

    To ask the right questions about the cohort and then answer them you need to understand what you’re dealing with in the first place. In theory each patient may have several slides, and each slide may contain several ROIs. And patients should have different properties, something to compare with something.<\/p>\n

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    The annotation to be grasped<\/div>\n<\/div>\n

    After studying the table it became clear which dimensions are important:<\/p>\n