Enrichment and mutation detection of circulating tumor cells from blood samples

The potential of circulating tumor cells (CTCs) in the diagnosis and prognosis of cancer patients has become increasingly attractive. However, molecular analysis of CTCs is hindered by low sensitivity and a high level of background leukocytes in CTC enrichment technologies. We have developed a novel protocol using a microfluidic device, which enriches and retrieves CTCs from blood samples. The principle of CTC capturing is that tumor cells are larger and less deformable than normal blood cells. To evaluate the potential of utilizing Celsee PREP100 in CTC molecular analysis, we prepared prostate cancer cell lines PC3 and LNCaP, retrieved the captured cells and analyzed them using PCR amplicon sequencing. We were able to recover an average of 79% of 110‑1,100 PC3 and 60‑1,500 LNCaP cells, and detect the p.K139fs*3 deletion of the p53 gene in PC3 cells and p.T877A mutation of the androgen receptor gene in LNCaP cells. Next, we spiked these two types of cells into normal donor blood samples, captured the cells and analyzed them using PCR amplicon sequencing. The PC3 and LNCaP cells were captured and retrieved with the ratio of captured CTCs to the background leukocytes reaching 1:1.5 for PC3 and 1:2.9 for LNCaP cells. We further revealed that the p.K139fs*3 deletion and p.T877A mutation can be detected in the captured PC3 and LNCaP cells, respectively. We successfully validated this approach using clinical blood samples from patients with metastatic prostate cancer. Our results demonstrated a novel approach for CTC enrichment and illustrated the potential of CTC molecular characterization for diagnosis, prognosis and treatment selection of patients with metastatic malignancy

Enrichment and mutation detection of circulating tumor cells from blood samples.

The potential of circulating tumor cells (CTCs) in the diagnosis and prognosis of cancer patients has become increasingly attractive. However, molecular analysis of CTCs is hindered by low sensitivity and a high level of background leukocytes in CTC enrichment technologies. We have developed a novel protocol using a microfluidic device, which enriches and retrieves CTCs from blood samples. The principle of CTC capturing is that tumor cells are larger and less deformable than normal blood cells. To evaluate the potential of utilizing Celsee PREP100 in CTC molecular analysis, we prepared prostate cancer cell lines PC3 and LNCaP, retrieved the captured cells and analyzed them using PCR amplicon sequencing. We were able to recover an average of 79% of 110‑1,100 PC3 and 60‑1,500 LNCaP cells, and detect the p.K139fs*3 deletion of the p53 gene in PC3 cells and p.T877A mutation of the androgen receptor gene in LNCaP cells. Next, we spiked these two types of cells into normal donor blood samples, captured the cells and analyzed them using PCR amplicon sequencing. The PC3 and LNCaP cells were captured and retrieved with the ratio of captured CTCs to the background leukocytes reaching 1:1.5 for PC3 and 1:2.9 for LNCaP cells. We further revealed that the p.K139fs*3 deletion and p.T877A mutation can be detected in the captured PC3 and LNCaP cells, respectively. We successfully validated this approach using clinical blood samples from patients with metastatic prostate cancer. Our results demonstrated a novel approach for CTC enrichment and illustrated the potential of CTC molecular characterization for diagnosis, prognosis and treatment selection of patients with metastatic malignancy

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Data from: Benefits and challenges with applying unique molecular identifiers in next generation sequencing to detect low frequency mutations

Indexing individual template molecules with a unique identifier (UID) before PCR and deep sequencing is promising for detecting low frequency mutations, as true mutations could be distinguished from PCR errors or sequencing errors based on consensus among reads sharing same index. In an effort to develop a robust assay to detect from urine low-abundant bladder cancer cells carrying well-documented mutations, we have tested the idea first on a set of mock templates, with wild type and known mutants mixed at defined ratios. We have measured the combined error rate for PCR and Illumina sequencing at each nucleotide position of three exons, and demonstrated the power of a UID in distinguishing and correcting errors. In addition, we have demonstrated that PCR sampling bias, rather than PCR errors, challenges the UID-deep sequencing method in faithfully detecting low frequency mutation

Epidemiological characteristics and spatial-temporal clusters of hand, foot, and mouth disease in Qingdao City, China, 2013-2018.

BACKGROUND:Hand, foot, and mouth disease (HFMD) has become one of the most important infectious diseases recent years. Qingdao City has suffered from serious HFMD epidemic. This study aimed to describe epidemiological characteristics and investigate spatial-temporal distribution at town level in Qingdao City. METHOD:The surveillance data of HFMD during 2013-2018 were collected from the National Notifiable Disease Surveillance System. The global Moran's I statistic was used to detect the spatial autocorrelation of HFMD cases by ArcGis 10.0 software. Purely spatial and spatial-temporal analysis was used to detect epidemic clusters by SatScanTM v9.6 software. RESULTS:The annual average incidence of HFMD cases in Qingdao City from 2013 to 2018 was 123.16 per 100000, while the incidence rate of children≤5years old was 2879.80 per 100000. The majority (88.97%) of HFMD cases were aged within 0-5 years old and the males were 60.20%. Other enterovirus (EV), enteriovirus 71(EV71), and Coxsackievirus A16 (CA16) accounted for 48.75%, 30.91% and 20.34%. The seasonal peak was between May and October. HFMD had positive spatial autocorrelation at town level with global Moran's I from 0.19 to 0.31(P<0.001). Spatial-temporal cluster analysis detected six most likely clusters and three secondary clusters from 2013 to 2018. The most likely cluster was located in urban and urban-rural fringe areas. CONCLUSIONS:Urban and urban-rural fringe areas were the major locations of the clusters with other EV as the dominant pathogen between May and October. The findings suggested that the prevention and control of HFMD in Qingdao City should be focus on these high-risk periods and locations which had important public health significance for the allocation of public health resources

7Mix_E7 (4)

4th part of 7Mix_E7 fastq dat

7Mix_E7 (3)

3rd part of the 7Mix_E7 fastq dat

7Mix E7 (2)

2nd part of 7Mix_E7 fastq dat

7Mix_E7 (1)

1st part of 7Mix_E7 fastq dat