June 1, 2021  |  

Highly sensitive, non-invasive detection of colorectal cancer mutations using single molecule, third generation sequencing.

Colorectal cancer (CRC) represents one of the most prevalent and lethal malignant neoplasms and every individual of age 50 and above should undergo regular CRC screening. Currently, the most effective procedure to detect adenomas, the precursors to CRC, is colonoscopy, which reduces CRC incidence by 80%. However, it is an invasive approach that is unpleasant for the patient, expensive, and poses some risk of complications such as colon perforation. A non-invasive screening approach with detection rates comparable to those of colonoscopy has not yet been established. The current study applies Pacific Biosciences third generation, single molecule sequencing to the inspection of CRC-driving mutations. Our approach combines the screening power and the extremely high accuracy of circular consensus (CCS) third generation sequencing with the non-invasiveness of using stool DNA to detect CRC-associated mutations present at extremely low frequencies and establishes a foundation for a non-invasive, highly sensitive assay to screen the population for CRC and early stage adenomas. We performed a series of experiments using a pool of fifteen amplicons covering the genes most frequently mutated in CRC (APC, Beta Catenin, KRAS, BRAF, and TP53), ensuring a theoretical screening coverage of over 97% for both CRC and adenomas. The assay was able to detect mutations in DNA isolated from stool samples from patients diagnosed with CRC at frequencies below 0.5 % with no false positives. The mutations were then confirmed by sequencing DNA isolated from the excised tumor samples. Our assay should be sensitive enough to allow the early identification of adenomatous polyps using stool DNA as analyte. In conclusion, we have developed an assay to detect mutations in the genes associated with CRC and adenomas using Pacific Biosciences RS Single Molecule, Real Time Circular Consensus Sequencing (SMRT-CCS). With no systematic bias and a much higher raw base-calling quality (CCS) compared to other sequencing methods, the assay was able to detect mutations in stool DNA at frequencies below 0.5 % with no false positives. This level of sensitivity should be sufficient to allow the detection of most adenomatous polyps using stool DNA as analyte, a feature that would make our approach the first non-invasive assay with a sensitivity comparable to that of colonoscopy and a strong candidate for the non-invasive preventive CRC screening of the general population.


April 21, 2020  |  

Divergent evolution in the genomes of closely related lacertids, Lacerta viridis and L. bilineata, and implications for speciation.

Lacerta viridis and Lacerta bilineata are sister species of European green lizards (eastern and western clades, respectively) that, until recently, were grouped together as the L. viridis complex. Genetic incompatibilities were observed between lacertid populations through crossing experiments, which led to the delineation of two separate species within the L. viridis complex. The population history of these sister species and processes driving divergence are unknown. We constructed the first high-quality de novo genome assemblies for both L. viridis and L. bilineata through Illumina and PacBio sequencing, with annotation support provided from transcriptome sequencing of several tissues. To estimate gene flow between the two species and identify factors involved in reproductive isolation, we studied their evolutionary history, identified genomic rearrangements, detected signatures of selection on non-coding RNA, and on protein-coding genes.Here we show that gene flow was primarily unidirectional from L. bilineata to L. viridis after their split at least 1.15 million years ago. We detected positive selection of the non-coding repertoire; mutations in transcription factors; accumulation of divergence through inversions; selection on genes involved in neural development, reproduction, and behavior, as well as in ultraviolet-response, possibly driven by sexual selection, whose contribution to reproductive isolation between these lacertid species needs to be further evaluated.The combination of short and long sequence reads resulted in one of the most complete lizard genome assemblies. The characterization of a diverse array of genomic features provided valuable insights into the demographic history of divergence among European green lizards, as well as key species differences, some of which are candidates that could have played a role in speciation. In addition, our study generated valuable genomic resources that can be used to address conservation-related issues in lacertids. © The Author(s) 2018. Published by Oxford University Press.


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