Single-Cell Sequencing Exposes Genetic Diversity in Acute Lymphoblastic Leukemia
Summary
The use of single-cell targeted DNA sequencing was employed in the investigation of T-cell acute lymphoblastic leukemia (T-ALL). Due to the ability to create a unique ALL panel, it was possible to accurately identify single-nucleotide variants and small insertion-deletion mutations in relation to 305 amplicons covering 110 genes, within an estimated 4400 cells per sample and time point, totaling 108 188 cells analyzed for 25 samples from 8 T-ALL patients. The study's findings showed that a major clone and multiple subsidiary clones were present at the time of diagnosis, and a sizable proportion of T-ALL cases had activating mutations in NOTCH1 and other genes involved in kinase signaling, transcriptional control, and protein translation. Remaining leukemic cells and minor clones were found in samples after longitudinal investigation, and these clones eventually developed into clinically relevant major clones at the late stages of the disease. According to the study, single-cell target DNA sequencing provides an extremely sensitive method for examining the clonal architecture and evolution of T-ALL.
Fig.1 Graphical abstract. (Albertí-Servera, 2021)
Research Criteria
In this study, the authors created a special library to screen for single-nucleotide variations (SNVs) and indels in 305 genomic areas, including the regions of 110 genes that frequently experience mutations in ALL. They genotyped over 4400 cells per sample simultaneously using droplet-based microfluidics technology, with great coverage to enable precise SNV and indel calling. Using paired T-ALL samples collected at diagnosis, therapy, and relapse, they used this.
Sample Type
Bone marrow (BM) and peripheral blood (PB) samples obtained at diagnosis, during treatment, and at relapse were collected from children diagnosed with T-ALL.
Result—A Novel Panel to Detect Gene Mutations at Single-Cell Level in ALL
To characterize the clonal architecture of T-ALL in primary patient samples, they performed single-cell DNA sequencing with the Tapestri platform. For the multiplex polymerase chain reaction amplification, they designed a custom panel that targets mutational hotspots in ALL. The panel consists of 305 amplicons with a median length of 192 bp that covers 110 genes. They sequenced 108,188 cells from 25 samples. They obtained a median throughput of 4444 cells per sample and a median sequencing coverage of 110 reads per amplicon per cell.
Fig.2 Custom ALL panel coverage and metrics of patient samples processed with the Tapestri platform for single-cell DNA sequencing. (Albertí-Servera, 2021)
Result—Single-Cell DNA Sequencing Reveals Similar Leukemic Clonal Composition in the BM and Blood
In a meticulous study of five patients diagnosed with T-cell acute lymphoblastic leukemia (T-ALL), the researchers aimed to comprehend the extent of clonal heterogeneity present in the blood and bone marrow (BM) of these patients. To achieve this objective, the researchers first compared the mutational profile of BM and blood from the patients and identified 100% of the variants in both tissues, with similar frequencies. Subsequently, the researchers delved deeper into the clonal architecture of the BM and blood samples to determine the final number and distribution of clones per patient, which was then depicted in the form of heatmaps, annotated with the origin of the cells. The analysis revealed that leukemia in BM and blood is largely similar, and there was little to no differential clonal selection of the leukemia cells in either tissue. Therefore, the results of the study demonstrate that molecular analysis of blood samples can be considered representative of the blasts in the BM, as the clonal frequencies fluctuate little between the two tissues. These findings provide important insights into the clonal heterogeneity of T-ALL patients, and the equivalence of leukemia in BM and blood serves as a crucial consideration for future molecular studies of this disease.
Fig.3 ALL has a similar mutational profile in the bone. (Albertí-Servera, 2021)
Creative Biolabs' Service
Single Cell DNA Sequencing Service
Creative Biolabs provides comprehensive whole genome amplification to discover DNA mutations in single cells. We examine genotype and phenotype from single cells at the same time, exposing the heterogeneity of your samples with unparalleled details. Single-cell DNA sequencing approach can be used to identify complete biomarkers that assist in better stratifying patients, detecting resistance earlier, and predicting recurrence.
Learn moreCreative Biolabs provides two types of sequencing workflow to meet the diverse needs of our customers. The first is microdroplet-based single cell panel-targeted DNA sequencing, which enables high-throughput analysis of specific gene regions of interest. This approach is ideal for exploring cell populations and uncovering genetic variation at the single cell level. The second workflow is plate-based single cell whole genome sequencing, which provides a comprehensive view of the entire genome of single cells. This workflow is ideal for applications that require a deep understanding of the genomic landscape of single cells, including genome-wide association studies and epigenetic profiling. Both workflows are highly reliable and are supported by our experienced technical team, who are dedicated to providing the highest quality data and analysis to our clients. For any information, please contact us.
Reference
- Albertí-Servera, L.; et al. Single-cell DNA amplicon sequencing reveals clonal heterogeneity and evolution in T-cell acute lymphoblastic leukemia. Blood. 2021, 137(6): 801-811.
