Week 7

Hi everyone!

I’ve had a very exciting week! While waiting for the sequencing results to come in, I got an introduction to several ongoing projects related to my own, focused on different approaches towards identifying the processes that are misregulated in myeloma cells.

The project I’ve had the most time to work on so far makes use of an analysis technique called mate pair sequencing, a powerful technique for identifying changes in the structure of chromosomes. Some of these structural variations (SVs) are already known to be major driving factors of oncogenesis, especially in multiple myeloma. SVs that bring oncogenes near enhancers or super-enhancers (DNA sequences that turn on nearby genes) are thought to have a major influence on how myeloma can progress at very different rates in patients. SVs involving a particular oncogene called Myc are a topic the Bergsagel lab has studied extensively using mate pair sequencing and other techniques. Currently, Myc translocations have been identified in the vast majority of myeloma patient samples and cell lines. Previous research shows that Myc translocations occur very late in tumor progression. Our working hypothesis is that Myc translocations might be what separates patients whose cancers never progress beyond a pre-cancerous stage called MGUS from patients who progress rapidly into full-blown MM.

To test this hypothesis, we need to search many patient samples taken at several timepoints for Myc translocations. Mate pair is excellent for screening the entire genome for SVs, but it’s expensive, slow, and requires relatively large quantities of very well-preserved patient samples. Analyzing mate pair data requires making some judgement calls on whether some ambiguous reads represent real SVs, so it can miss certain SVs. Instead, by analyzing Myc expression directly, we can more precisely identify samples with Myc translocations. Because IgL rearrangements are generated randomly in pre-B cells, each IgL rearrangement will be unique to a particular lineage of plasma cells (called a clone). In any single patient, the myeloma/MGUS lesions are not monoclonal, meaning a single tumor sample actually consists of a mix of different clones. The qPCR assays I’m designing will let us quantify the proportion of a sample consisting of a particular clone, letting us compare Myc expression levels to the relative abundance of the clone in question.

On Thursday, the sequencing results came in. Although the sequencing data was excellent in terms of clarity, the primer set I had used didn’t cover enough of the J end of the VJ rearrangements, meaning we can’t generate the qPCR assays just yet. To fix this, I ordered new J primers that anneal further downstream, meaning the amplicon should be long enough to determine the entire full sequence of the VJ rearrangement. Hopefully, by next Thursday we’ll have the new sequencing results, and from these I’ll be able to design an initial qPCR assay!

Thanks for reading!