SARS-CoV-2 genome sequencing with Oxford Nanopore Technology and Rapid PCR Barcoding in Bolivia

SARS-CoV-2 genomic surveillance has Illumina technology as the golden standard. However, Oxford Nanopore Technology (ONT) provides significant improvements in accessibility, turnaround time and portability. Characteristics that gives developing countries the opportunity to perform genome surveillance. The most used protocol to sequence SARS-CoV-2 with ONT is an amplicon-sequencing protocol provided by the ARTIC Network which requires DNA ligation. Ligation reagents can be difficult to obtain in countries like Bolivia. Thus, here we provide an alternative for library preparation using the rapid PCR barcoding kit (ONT). We mapped more than 3.9 million sequence reads that allowed us to sequence twelve SARS-CoV-2 genomes from three different Bolivian cities. The average sequencing depth was 324X and the average genome length was 29527 bp. Thus, we could cover in average a 98,7% of the reference genome. The twelve genomes were successfully assigned to four different nextstrain clades (20A, 20B, 20E and 20G) and we could observe two main lineages of SARS-CoV-2 circulating in Bolivia. Therefore, this alternative library preparation for SARS-CoV-2 genome sequencing is effective to identify SARS-CoV-2 variants with high accuracy and without the need of DNA ligation. Hence, providing another tool to perform SARS-CoV-2 genome surveillance in developing countries.

Human whole genome sequencing in South Africa

The advent and evolution of next generation sequencing has considerably impacted genomic research. Until recently, South African researchers were unable to access affordable platforms capable of human whole genome sequencing locally and DNA samples had to be exported. Here we report the whole genome sequences of the first six human DNA samples sequenced and analysed at the South African Medical Research Council’s Genomics Centre. We demonstrate that the data obtained is of high quality, with an average sequencing depth of 36.41, and that the output is comparable to data generated internationally on a similar platform. The Genomics Centre creates an environment where African researchers are able to access world class facilities, increasing local capacity to sequence whole genomes as well as store and analyse the data.

Influence of Inherited and Acquired Hematological and Immunological Gene Variants on the Outcomes of Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Hematological Malignancies

Chromosome abnormalities and gene variants are important factors in prognosis of the patients with hematological malignancies. Our previous study showed that some germline gene mutations-related to hematological and immunological disorders may have negative impact on the complications of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In current study, the influence of both hematological and immunological hereditary predisposition gene variants and tumor gene variants on the outcomes of allo-HSCT in patients with hematological malignancies was studied. Between January 2018 and June 2020, 164 patients with hematological malignancies who underwent allo-HSCT in our hospital were analyzed. The median age was 14 (1 to 67) years old. The diagnosis included acute myeloid leukemia (n=68, 41.5%), acute lymphoblastic leukemia (n=67, 40.8%) and non-Hodgkin's lymphoma (n=29, 17.7%). The disease status before transplant was CR1 in 49 cases (29.9%), CR2 in 63 cases (38.4%), PR in 22 cases (13.4%), and NR in 30 cases (18.3%). Donors were from haploidentical family members (n=125, 76.2%) or identical siblings (n=18, 11.0%) or unrelated volunteers (n=21, 12.8%). Myeloablative conditioning regimens with either total body irradiation/fludarabine-based or busulfan/fludarabine-based were applied. Anti-thymocyte globulin was used in haploidentical and unrelated transplants. Graft-versus-host disease (GVHD) prophylaxis was with cyclosporine, short-term methotrexate and mycophenolate mofetil. Before transplant, blood samples from patients, their parents and potential related donors were collected to analyze for more than 700 kinds of hematological and immunological hereditary predisposition genes with whole exon sequencing and validation by sanger sequencing. The average sequencing depth was 150×. At diagnosis or relapse, bone marrow samples from patients were obtained to detect for 339 kinds of tumor genes by Illumina sequencing. The average sequencing depth was more than 3000×. With the median follow-up 12.6 (11.2 to 17.0) months, 105 patients (64.0%) achieved durable remission after allo-HSCT. Forty-seven patients (28.7%) relapsed. Twelve patients (7.3%) died. Total 191 immunodeficiency-related hereditary predisposition gene variants were identified in this cohort (average 3.5 gene variants per patient; range, 0-10). Twenty-six of them were recurrent more than 6 times that including TYK2, IFIH1, CFTR, LRBA, IL7R, POLE, RNF31, NLRP12, TTC7A, ATM, CARD14, CHD7, NOD2, TNFRSF13B, BLB,CFB, EPG5, C8A, C8B, CFH, IRF, MSH6, NCF2, NFAT5, PMS2 and ST1M1.IFIH1and IL10RB gene variants were poor factors for relapse post-transplant (P=0.006, P=0.007). The functions of these genes involve in combined immunodeficiency, autoinflammatory disease, complement deficiency, immune deficiency, T-cell dysfunction and antibody deficiency. Total 153 tumor gene variants were identified in this patient series (average 1.93 gene variants per patient; range, 0-16). Seventeen of them were recurrent more than 4 times that including TP53, TPN11, KIT, FLT3, NRAS, NUDT15, STK11, CREBBP, NPM1, DNAH9, DNMT3A, IDH2, HEK2, KRAS, KMT2C, NOTCH1 and NR3C1. TP53, KRAS and NUDT15 gene variants were poor factors for relapse after allo-HSCT (P=0.000025, P=0.0082, P=0.000018). Hemophagocytic lymphohistiocytosis (HLH)-related genes variants were found in 9 patients (5.5%). Frequent HLH-related gene variants were in CD27, PRF1, STX11, and UNC13D. Fanconi anemia (FA)-related gene variants were seen in 11 patients (6.7%). Common FA-related gene variants were in BRCA1, BRCA2, BRIP1, FANCA, FANCC, FANCF, FANCM and FANCG. Significantly higher incidences of acute GVHD (aGVHD) and/or infections were noted in the patients with HLH and/or FA-related gene variants compared with those without HLH and FA-related gene variants (p=0.019). Our results have shown that both inherited and acquired hematological and immunological-related gene variant profiles in the patients with hematological malignancies and some recurrent gene variants (IFIH1 and IL10RB; TP53, KRAS and NUDT15) have negative impact on the outcomes of allo-HSCT including leukemia-free survival, aGVHD and infections. Keywords: allogeneic hematopoietic stem cell transplant, gene expression, relapse, hematological malignancy Disclosures No relevant conflicts of interest to declare.

Human Whole Genome Sequencing in South Africa

ABSTRACTThe advent and evolution of next generation sequencing has considerably impacted genomic research. Until recently, South African researchers were unable to access affordable platforms capable of human whole genome sequencing locally and DNA samples had to be exported. Here we report the whole genome sequences of the first six human DNA samples sequenced and analysed at the South African Medical Research Council’s Genomics Centre. We demonstrate that the data obtained is of high quality, with an average sequencing depth of 36.41, and that the output is comparable to data generated internationally on a similar platform. The Genomics Centre creates an environment where African researchers are able to access world class facilities, increasing local capacity to sequence whole genomes as well as store and analyse the data.

CIC-Mutation As a Potential Molecular Mechanism of Acquired Resistance to Combined BRAF/MEK Inhibition in CNS Multiple Myeloma

Central nervous system (CNS) involvement is an extremely rare extramedullary multiple myeloma (MM) manifestation, diagnosed in less than 1% of patients. It is considered an ultimate high-risk feature, associated with unfavorable cytogenetics, and, even with intense treatment applied, survival is short, reaching less than 12 months in most cases. In June 2017 an 81 years old male with a κ light chain MM was referred to our institution for an isolated CNS MM relapse. His cerebrospinal fluid (CSF) demonstrated a high load of clonal plasma cells, however, the patient's bone marrow infiltration was very little with a percentage of plasma cells less than 5%. Imaging, including gold standard MRI and experimental 11C-methionine PET scan, was performed, and high metabolic activity was detected supra- and infratentorially as well as in the right femur and the clivus. Following CD138+ cell purification we analyzed the specimen with M3P (v3.0) a disease specific in-house customized, next generation targeted sequencing panel for MM (Ion torrent platform). This includes most commonly mutated MM genes, actionable drug targets and drug resistance associated genes. The average sequencing depth increased 700X and spatial MM heterogeneity was detected, as the CFS cells harbored a clonal BRAFV600E mutation, absent in the bone marrow. Initial intrathecal and systemic chemotherapy with Cytarabine and Thiotepa was intolerable, thus the patient underwent a combined target inhibition with Dabrafenib/Trametinib, well known specific BRAF and a MEK 1/2 inhibitors. The patient displayed a rapid complete response (Figure. 1A), however, disease relapse occurred after three months of therapy. We obtained a sequential CFS sample and Whole Exome Sequencing (Illumina platform) was applied to pre and post therapy CFS sampling. Exome sequencing of the two time points performed an average sequencing depth of 115X; a total number of 97 non-silent coding variants (missense, nonsense, indels, splice) with an allele frequency higher than 5% were detected. In detail, 19 point mutations were acquired at relapse, including a subclonal missense mutation in CIC (p.A984P, VRF 17%), recently identified as a candidate gene contributing to MEK/BRAF resistance development. Next, we established a CIC knock-down model electroporating a specific anti-CIC siRNA into U266 MM cell line. We cultured the silenced and not-silenced cells with Trametinib and Dabrafenib, either as single agents, or in combination. As expected, we observed resistance induction to the combination of the two drugs (Row Factor 85.94%; P<0.0001, Two-way ANOVA) suggesting a critical role for this patient derived mutation for his MEK/BRAF resistance development (Figure 1C, D). In order to better clarify the landscape pathway related to CIC we analyzed expression data from 647 patients enrolled in the MMRF CoMMpass trial. Remarkably, we found a significant down-regulation of ERF and ETV6 (t-test -9.95, -9.93, P <0.001, respectively), two well characterized tumor suppressor genes correlated with the re-activation of the RAS downstream pathway (Figure 1B). This is the first report giving evidence for a potential role of point mutations in CIC as a resistance mechanism to targeted MEK/BRAF inhibition in BRAF mutated MM. The performed pathway analysis significantly extends the insights of the resistance mechanisms highlighted. Our results foster a statistically powered study to corroborate the clinical relevance. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Development and performance of a targeted whole exome sequencing enrichment kit for the dog (Canis Familiaris Build 3.1)

Whole exome sequencing is a technique that aims to selectively sequence all exons of protein-coding genes. A canine whole exome sequencing enrichment kit was designed based on the latest canine reference genome (build 3.1.72). Its performance was tested by sequencing 2 exome captures, each consisting of 4 pre-capture pooled, barcoded Illumina libraries on an Illumina HiSeq 2500. At an average sequencing depth of 102x, 83 to 86% of the target regions were completely sequenced with a minimum coverage of five and 90% of the reads mapped on the target regions. Additionally, it is shown that the reproducibility within and between captures is high and that pooling four samples per capture is a valid option. Overall, we have demonstrated the strong performance of this WES enrichment kit and are confident it will be a valuable tool in future disease association studies.