Large Amplicons Research Articles

The first large scale rDNA amplicon database of soil microbiomes from Pamir Plateau, China

The Eastern Pamir, distinguished with high altitude, extremely arid and cold climate, limited nutrients and sparse vegetation, is a unique ecological reservoir. Microbial communities play a central role in maintaining Eastern Pamir’s ecosystem functioning. Despite the ecological significance, due to the difficulty of sample collection and microbial isolation, the microbial diversity and its functionality at the Pamir Plateau have been rarely documented. To fill this gap, 80 soil samples from 17 sites across different elevations were collected, performed the rDNA amplicon sequencing to present the first large-scale overview of bacterial, archaeal, and fungal communities in the Eastern Pamir. Microbiome analysis revealed that the bacteria Actinobacteria, Alphaproteobacteria and Bacteroidia, alongside such as archaea Nitrososphaeria and Halobacteria, and fungi including Dothideomycetes, Sordariomycetes and Eurotiomycetes were dominant lineages at class level in soil microbial communities. The community structure and biodiversity of soil microorganisms provided by this dataset would be pivotal for future studies aimed at understanding the biogeographical distribution, ecological functions and environmental responses of microbial communities of the Pamir Plateau.

CRISPR-Cas guide RNA indel analysis using CRISPResso2 with Nanopore sequencing data

ObjectiveInsertion and deletion (indel) analysis of CRISPR-Cas guide RNAs (gRNAs) is crucial in gene editing to assess gRNA efficiency and indel frequency. This study evaluates the utility of CRISPResso2 with Oxford Nanopore sequencing data (nCRISPResso2) for gRNA indel screening, compared to two common Sanger sequencing-based methods, TIDE and ICE. To achieve this, sheep and horse fibroblasts were transfected with Cas9 and a gRNA targeting the myostatin (MSTN) gene. DNA was subsequently extracted, and PCR products exceeding 600 bp were sequenced using both Sanger and Nanopore sequencing. Indel profiling was then conducted using TIDE, ICE, and nCRISPResso2.ResultsComparison revealed close correspondence in indel formation among methods. For the sheep MSTN gRNA, indel percentages were 52%, 58%, and 64% for TIDE, ICE, and nCRISPResso2, respectively. Horse MSTN gRNA showed 81%, 87%, and 86% edited amplicons for TIDE, ICE, and nCRISPResso2. The frequency of each type of indel was also comparable among the three methods, with nCRISPResso2 and ICE aligning the closest. nCRISPResso2 offers a viable alternative for CRISPR-Cas gRNA indel screening, especially with large amplicons unsuitable for Illumina sequencing. CRISPResso2’s compatibility with Nanopore data enables cost-effective and efficient indel profiling, yielding results comparable to common Sanger sequencing-based methods.

Comparative evaluation of 16S rRNA primer pairs in identifying nitrifying guilds in soils under long-term organic fertilization and water management.

Compared with 454 sequencing technology, short-read sequencing (e.g., Illumina) technology generates sequences of high accuracy, but limited length (<500 bp). Such a limitation can prove that studying a target gene using a large amplicon (>500 bp) is challenging. The ammonia monooxygenase subunit A (amoA) gene of ammonia-oxidizing archaea (AOA), which plays a crucial part in the nitrification process, is such a gene. By providing a full overview of the community of a functional microbial guild, 16S ribosomal ribonucleic acid (rRNA) gene sequencing could overcome this problem. However, it remains unclear how 16S rRNA primer selection influences the quantification of relative abundance and the identification of community composition of nitrifiers, especially AOA. In the present study, a comparison was made between the performance of primer pairs 338F-806R, 515F-806R, and 515F-907R to a shotgun metagenome approach. The structure of nitrifier communities subjected to different long-term organic matter amendment and water management protocols was assessed. Overall, we observed higher Chao1 richness diversity of soil total bacteria by using 515F-806R compared to 338F-806R and 515F-907R, while higher Pielou's evenness diversity was observed by using 515F-806R and 515F-907R compared to 338F-806R. The studied primer pairs revealed different performances on the relative abundance of Thaumarchaeota, AOB, and NOB. The Thaumarchaeota 16S rRNA sequence was rarely detected using 338F-806R, while the relative abundances of Thaumarchaeota detected using 515F-806R were higher than those detected by using 515F-907R. AOB showed higher proportions in the 338F-806R and 515F-907R data, than in 515F-806R data. Different primers pairs showed significant change in relative proportion of NOB. Nonetheless, we found consistent patterns of the phylotype distribution of nitrifiers in different treatments. Nitrosopumilales (NP) and Nitrososphaerales (NS) clades were the dominant members of the AOA community in soils subject to controlled irrigation, whereas Ca. Nitrosotaleales (NT) and NS clades dominated the AOA community in soils subject to flooding irrigation. Nitrospira lineage II was the dominant NOB phylotype in all samples. Overall, ideal 16S rRNA primer pairs were identified for the analysis of nitrifier communities. Moreover, NP and NT clades of AOA might have distinct environmental adaptation strategies under different irrigation treatments.

AMPLON: Amplifying DNA with Multiarm Priming and Looping Optimization of Nucleic Acid.

Nucleic acid amplification, the bedrock of biotechnology and molecular diagnostics, surges in applications-especially isothermal approaches-heightening the demand for advanced and precisely engineered methods. Here, a novel approach for amplifying DNA with multiarm priming and looping optimization of nucleic acid (AMPLON) is presented. AMPLON relies on a novel polymeric material with unique set of multiarm polyethylene glycol-DNA primers for efficient DNA amplification under isothermal conditions. Each arm carries single-stranded DNA complementing the sense or antisense sequence of the target DNA. The amplification reaction begins with antisense arms binding to the target DNA, forming a template for sense-carrying arms to direct multiarm large DNA amplicon synthesis through successive DNA looping and unlooping steps. Using human immunodeficiency virus type 1 (HIV-1) as a model clinical target, AMPLON exhibits high sensitivity, detecting target concentrations as low as 100 copies mL-1. Compared to a quantitative real-time polymerase chain reaction assay using sensitive primers, AMPLON reliably identifies HIV-1 RNA in plasma samples (n = 20) with a significant agreement rate of 95%. With its ability to achieve highly specific and sensitive target amplification within 30 min, AMPLON holds immense potential to transform the field of nucleic acid research and unleashing new possibilities in medicine and biotechnology.

Development and validation of a new multiplex panel using SNaPshot-based DIP-TriSNP markers for forensic DNA mixtures.

Short tandem repeat analysis is challenging when dealing with unbalanced mixtures in forensic cases due to the presence of stutter peaks and large amplicons. In this research, we propose a novel genetic marker called DIP-TriSNP, which combines deletion/insertion polymorphism (DIP) with tri-allelic single nucleotide polymorphism in less than 230bp length of human genome. Based on multiplex PCR and SNaPShot, a panel, including 14 autosomal DIP-TriSNPs and one Y chromosomal DIP-SNP, had been developed and applied to genotyping 102 unrelated Han Chinese individuals in Sichuan of China and simulated a mixture study. The panel sensitivity can reach as low as 0.1ng DNA template, and the minor contributor of DNA can be detected with the highest ratio of 19:1, as indicated by the obtained results. In the Sichuan Han population, the cumulative probability of informative genotypes reached 0.997092, with a combined power of discrimination of 0.999999998801. The panel was estimated to detect more than two alleles in at least one locus in 99.69% of mixtures of the Sichuan Han population. In conclusion, DIP-TriSNPs have shown promising as an innovative DNA marker for identifying the minor contributor in unbalanced DNA mixtures, offering advantages such as short amplifications, increased polymorphism, and heightened sensitivity.

Characterization of DNA Degrading Microorganisms from Dewar Creek Hot Springs in Western Canada

Historically, discovery and subsequent characterization of microbial species relied on pure cultures. Some challenges associated with creating pure cultures have been overcome with advances in culture independent and DNA-based molecular methods such as single-cell genomics, metagenomics, or large scale amplicon sequencing. With these advances, the rate of discovery of new species from genomic data has quickly outpaced the number of organisms with cultured representatives. As description and characterization still rely on cultures, our understanding of yet uncultured species is greatly lacking. Major lineages in the bacterial domain equivalent to phyla that lack any cultured representatives are termed “candidate phyla.” Candidate phyla are found across the bacterial tree of life, and many uncultured organisms are found to be dominant in understudied environments. Extreme environments such as thermal springs are an example of understudied environments, making them excellent environments for studying novel microbial lineages. The objective of this research is to characterize uncultured bacterial lineages from Dewar Creek hot spring in Western Canada, with a focus on DNA and protein metabolizing bacteria. Based on previous genomic data from organisms in this hot spring, we hypothesize that the candidate phylum S2R-29, with extremely low GC content, metabolizes extracellular DNA or protein Dewar Creek is a thermal spring located in the Purcell Wilderness Conservancy in British Colombia, Western Canada. It is one of Canada’s hottest springs, reaching temperatures of up to 83˚C. Samples of DNA extracted from the Dewar Creek hot spring were PCR-amplified with primers for the v3v4 region of the 16S rRNA gene to detect S2R-29, and then sequenced on an Illumina Miseq. S2R-29 was found in samples ranging in temperature from 60˚C to 77 ˚C. In addition to samples from Dewar Creek, samples from other thermal springs in Canada, as well as samples from springs in New Zealand are also being sequenced with the same primers to determine the prevalence of this candidate phylum in other similar environments. This will give a better idea of the growth conditions and range of this organism. Previously, S2R-29 single amplified genomes (SAGs) were generated from Dewar Creek samples. Analysis of these SAGs suggests that S2R-29 has the potential to use peptides and DNA as carbon sources (Fig. 1). To test the potential to metabolize DNA and protein, enrichments of samples with 13C labelled dNTPs or 13C labelled protein have been started. These enrichments will be used for stable isotope probing (SIP) to determine if any organisms in Dewar Creek are metabolizing dNTPs or protein. In addition to SIP, primers specific for S2R-29 for quantitative PCR (qPCR) have been designed and will be run on the enrichments to determine if there are any changes in the abundance of S2R-29 over time in these enrichments, further testing the metabolic potential of these organisms. Finally, probes for fluorescence in situ hybridization (FISH) will be designed for S2R-29. These will be used to perform FISH with samples from Dewar Creek in order to visualize this candidate phylum. With this research, we hope to characterize this novel phylum, and possibly discover other novel lineages of DNA- and protein- metabolizing bacteria in Dewar Creek. This research will help better understand these processes in understudied environments as well as aid our understanding of the roles that bacterial metabolisms might play in biogeochemical cycles such as biodegradation of organic matter. In all, we hope that this research gives insight into the kinds of lifestyles bacteria have evolved in order to thrive in high temperature environments.

A supplementary analysis of a large scale 16S ribosomal RNA gene amplicon dataset of hand, foot and mouth patients and healthy individuals

A supplementary analysis of a large scale 16S ribosomal RNA gene amplicon dataset of hand, foot and mouth patients and healthy individuals

Haplotype Disequilibrium in the TLR Genes of Czech Red Pied Cattle

Hybrid resequencing of the antibacterial innate immune genes coding for toll-like receptors, namely TLR1, TLR2, TLR4, TLR5, and TLR6, using HiSeq and PacBio technologies of pooled population samples of Czech Simmental (Czech Red) cattle allowed us to determine haplotypes formed by the polymorphisms present. Directly determined haplotypes within the range of the large proximal amplicon in TLR2 formed two clusters in the network tree graph. The distribution of the statistically reconstructed haplotypes based on individual genotyping of the present SNPs was consistent. Similarly, the statistically reconstructed haplotypes in TLR5 and TLR6 formed two clusters. The trend of bimodal distribution was also observed in TLR4, while the limited diversity of TLR1 did not allow for any conclusion. The observed bimodal distribution is consistent with earlier reports for cattle populations worldwide. The stability of this phenomenon cannot be ascribed to historical origin but rather to a long-term effect of balancing selection. The equilibrium might be based on two different essential functions performed by the TLR genes or their products. The formation of two kinds of heterodimers by the TLR2 product, namely, TLR2/TLR1 and TLR2/TLR6 with different ligand specificities, is considered to be a particular case. On the other hand, the better expression of the bimodal groups in the 5′-proximal SNPs supports the localization of the selection targets in the upstream regulatory regions or the functional interactions in the proximal part of the transcripts.

Prognostic relevance of 3q amplification (AMP) in a racially diverse patient population with advanced squamous cell carcinoma of the lung.

e15142 Background: Amplification (AMP) of 3q is the most common genetic alteration in squamous cell carcinoma of the lung (LUSC) with the most frequently amplified region being 3q26-3q28. It represents one of the most striking molecular differences between LUSC and adenocarcinoma. We identified a minimal common region (MCR) of AMP constituted by 25 genes within a large amplicon on chromosome 3q by analyzing data of 476 patients with LUSC from The Cancer Genome Atlas (TCGA) and extracting copy number alteration data from cBioPortal. Full 25-gene MCR AMP was detected in 46.4% of the cases and was associated with better disease-specific survival (NR vs 9.25 years, 95% CI [5.24-NR]; p = 0.011) and progression-free interval (8 vs 4.9 years, 95% [3.5-NR]; p = 0.020). The TCGA cohort was mainly comprised of Caucasians (69.7%) and patients with early-stage disease (98.7%). We conducted a retrospective pilot analysis to validate the prognostic value of MCR AMP using a racially diverse cohort with advanced LUSC. Methods: We retrieved tissue samples for 33 patients with stage III or stage IV LUSC and collected retrospective data from Electronic Medical Records. The pathologist reviewed the slides to ensure the adequacy of tumor tissue. MCR AMP was evaluated via laboratory- developed FISH probe spanning MCR region. FISH test that showed a cluster of signals with more than 4 copies was regarded as positive; we used a control probe for the 3p arm which is typically not amplified in LUSC. Results: The mean age of the cohort was 69.1 years, 57.6% were males, 39.4% were African Americans (AA) and 54.5% were Caucasians, 66.7% had stage 3, and 33.3% had stage 4. Only 6.1% were never smokers and 84.8% were former smokers. MCR AMP was detected in 54.5% (n = 18) of the cases. PD-L1 status was available for 21 (63%) cases, 6 cases (3 had MCR AMP) were PD-L1 negative (TPS &lt; 1%), and 15 cases (9 had MCR AMP) were PD-L1 positive (TPS &gt; 1%). Consistent with the results observed in the TCGA cohort, MCR amplified cases had a trend towards better overall survival (OS) compared to non-amplified cases (1.74 vs 0.91 years; HR = 0.57, 95% CI [0.26-1.24], p = 0.15) as well as a trend towards better progression-free survival (PFS) (0.90 vs 0.74 years; HR = 0.88, 95% CI [0.43-1.81], p = 0.73). Conclusions: This pilot analysis shows that MCR AMP carries a prognostic value in a racially diverse patient population with advanced LUSC, similar to our observations in the TCGA cohort. Further validation in a large cohort is in progress.

A large scale 16S ribosomal RNA gene amplicon dataset of hand, foot and mouth patients and healthy individuals

There is evidence linking hand, foot and mouth disease (HFMD) to gut microbiota dysbiosis, and this relationship was corroborated in a large HFMD patient population in our previous study. Here, we present a bacterial 16S rRNA gene dataset from faecal samples of 713 individuals (254 HFMD patients, 459 healthy controls) aged 2 to 7 years residing in Heyuan and Jiangmen counties, Guangdong Province, southern China. Microbiome analysis indicated a significant increase in genus Prevotella, Cetobacterium, and Megamonas was observed in patients with HFMD, whereas a large increase in genus Bacteroides, Ruminococcus, and Faecalibacterium were seen in the control group. We also share the bioinformatic analytical pipeline for this analysis, from data preprocessing to data filtering and amplicon sequence variant (ASV) table generation. We expect that the dataset will be reprocessed, evaluated and fully analysed with various analysis methods to further elucidate the role of the gut microbiota in HFMD development.

Forensic Validation Studies of a Novel 35-InDel Multiplex Polymerase Chain Reaction System

Abstract Background: A difficulty associated with forensic applications is the detection of degraded biological materials. Due to the large amplicon sizes of short tandem repeat alleles, valid genotyping results cannot be obtained from degraded biological materials. Recently, insertion/deletion (InDel) polymorphisms have been used in forensic applications for their widespread distributions in the human genome, short amplicon sizes, and low mutation rates. Purpose: Human identification InDel panels have mostly been designed for European populations. Therefore, our laboratory independently developed a multiplex polymerase chain reaction (PCR) system with 35 polymorphic InDel loci to be used for human identification in China. Forensic validation studies were conducted on this novel 35-InDel multiplex PCR system. Methods: The 35 InDel loci were screened in the database, and then used with the traditional PCR amplification and capillary electrophoresis platform combined with five-color fluorescence parallel detection technology. Validation studies were performed on this novel panel, including accuracy, repeatability and reproducibility, species specificity, sensitivity, stability, forensic case sample detection, and mixture studies. In addition, forensic efficiency assessments were conducted in populations from different continents. Results: The data of validated studies indicated that the novel 35-InDel panel was accurate, stable, and efficient for forensic purposes. For human identification, the cumulative power of discrimination values for the these 35 InDel loci in East Asian, South Asian, European, American, and African populations were 0.999999999999995, 0.999999999999995, 0.999999999999971, 0.9999999999999960, and 0.999999999998166, respectively. Conclusions: In this study, a set of 35 InDel loci were conducted in a multiplex amplification system for human identification of degraded DNA sample, and this new assay was efficient and stable. The present results suggested that the 35-InDel panel was a reliable tool for forensic use and could be efficiently used for human identification in the East Asian populations.

Tracing the origin of an imported HIV-1 sub-subtype A6 strain first identified in Zhejiang Province, China.

Human migration and mobility have been identified as key drivers of HIV dissemination among nations, which increases the problem of genetic diversity. Here, we report the near full-length genome of HIV-1 A6 identified in a female patient in the remote mountain area of Lishui, Zhejiang Province, which is the first time A6 has been reported in China. The near full-length genome was amplified with two large amplicons of 5.5kb and 3.7kb, and then target PCR products were sequenced by Sanger sequencing. The A6 strain was confirmed by the Basic Local Alignment Search Tool (BLAST) and a maximum-likelihood (ML) phylogenetic tree. The time to the most recent common ancestor (tMRCA) was inferred to be 2004 (95% HPD interval: 2003-2006). The sequence harbored the L74I mutation, which is a key characteristic genetic marker of A6. Combining the above evidence with epidemiological investigations, this A6 strain was determined to be from Ukraine, which was supported by phylogenetic analysis. This study identified a foreign imported strain, indicating a trend of increasing complication in the HIV-1 epidemic in Zhejiang, China.

A novel set of short microhaplotypes based on non-binary SNPs for forensic challenging samples.

Short tandem repeats (STRs) are the most widely used genetic markers in forensic application, but they are not ideal genetic markers for the analysis of forensic challenging samples such as highly degraded or unbalanced mixed samples because of their relatively large amplicons and stutter peaks. In this study, we developed a set of short microhaplotypes based on non-binary SNPs with molecular extent sizes no longer than 60 bases and genotyped 100 unrelated individuals from northern Han groups. Our results showed this panel has similar discrimination power to STR kits, as the combined random match probability (CMP) reached 1.396 × 10-22 and mean effective number of alleles (Ae) was 3.59. The cumulative probability of exclusion for duos (CPE-duos) was 0.999919 and the cumulative probability of exclusion for trios (CPE-trios) was 0.9999999987, suggesting this panel could be applied for forensic personal identification and parentage testing independently. Population differentiation in 26 populations from the 1000 Genomes Project indicated this panel could distinguish populations from Africa, East Asia, South Asia, America, and Europe. These microhaplotypes based on non-binary SNPs have short amplicons, good discrimination power, no stutter artifacts, and have great potential in detection of highly degraded and unbalanced mixtures for personal identification, paternity testing, and ancestry inference.

Concatenation of paired-end reads improves taxonomic classification of amplicons for profiling microbial communities

BackgroundTaxonomic classification of genetic markers for microbiome analysis is affected by the numerous choices made from sample preparation to bioinformatics analysis. Paired-end read merging is routinely used to capture the entire amplicon sequence when the read ends overlap. However, the exclusion of unmerged reads from further analysis can result in underestimating the diversity in the sequenced microbial community and is influenced by bioinformatic processes such as read trimming and the choice of reference database. A potential solution to overcome this is to concatenate (join) reads that do not overlap and keep them for taxonomic classification. The use of concatenated reads can outperform taxonomic recovery from single-end reads, but it remains unclear how their performance compares to merged reads. Using various sequenced mock communities with different amplicons, read length, read depth, taxonomic composition, and sequence quality, we tested how merging and concatenating reads performed for genus recall and precision in bioinformatic pipelines combining different parameters for read trimming and taxonomic classification using different reference databases.ResultsThe addition of concatenated reads to merged reads always increased pipeline performance. The top two performing pipelines both included read concatenation, with variable strengths depending on the mock community. The pipeline that combined merged and concatenated reads that were quality-trimmed performed best for mock communities with larger amplicons and higher average quality sequences. The pipeline that used length-trimmed concatenated reads outperformed quality trimming in mock communities with lower quality sequences but lost a significant amount of input sequences for taxonomic classification during processing. Genus level classification was more accurate using the SILVA reference database compared to Greengenes.ConclusionsMerged sequences with the addition of concatenated sequences that were unable to be merged increased performance of taxonomic classifications. This was especially beneficial in mock communities with larger amplicons. We have shown for the first time, using an in-depth comparison of pipelines containing merged vs concatenated reads combined with different trimming parameters and reference databases, the potential advantages of concatenating sequences in improving resolution in microbiome investigations.

37 Genomic improvement of the horse X chromosome and characterization of the pseudoautosomal boundary

The horse reference genome has been improved with the release of EquCab3 and the first Y chromosome reference. The most complex sequences, such as those in the sex chromosomes, are still unresolved. These include large amplicons, repeats, and the pseudoautosomal boundary (PAB). We initiated a comprehensive study specifically to improve the assembly of the horse sex chromosomes. To refine the assembly of complex regions, we are utilizing 3 new technologies: trio-binning, Hi-C and Bionano optical mapping. Trio-binning uses long read sequences from F1 interspecific hybrids and short reads from parent species. High molecular weight blood DNA was extracted from a female hinny and sequenced on 2 PacBio Sequel cells. Paired-end Illumina reads (150bp) for horse ( Twilight ) and donkey ( Willy ) were obtained from SRA. These sequences as well as the hinny long reads were assembled with trio-binning function of the Canu assembler program. The initial assembly is 2.5 Gb separated into 1,757 contigs with an N50 of 41.5 Mb. We have completed Hi-C sequencing and Bionano optical maps for one thoroughbred stallion ( Bravo ). Both technologies are needed to scaffold the trio-binning Canu assembly. Our initial goal was to use this assembly to better define the PAB in the horse. The PAB demarcates the end of the pseudoautosomal region (PAR) where X-Y recombination stops. Despite the evolutionary and biological importance of the PAB, the region has been characterized at molecular level in only a few species. The PAB of the horse is presently not well defined. Previously, we identified and Sanger sequenced 4 BAC clones - 2 spanning PAB-X and PAB-Y. To identify the PAB of the horse, BAC sequences were aligned to the Y assembly, EquCab3 and a 42 Mb-size contig from trio-binning assembly which corresponds to the short arm of the X. We identified a region on both X and Y where X-Y homology drops from over 97% (PAR) to almost zero, indicative of the PAB. This region corresponds to the location of the XKR3Y gene in the Y but is not well-annotated in the X. We also identified a duplication and an inversion in EquCab3 which was not consistent with the corresponding region in the X-BACs, or the new 42 Mb Xp contig, suggesting a mis-assembly in EquCab3. We believe that these approaches combined will also resolve other complex and repetitive portions in the horse sex chromosomes.

Feasibility of Cell-Free DNA Collection and Clonal Immunoglobulin Sequencing in South African Patients With HIV-Associated Lymphoma.

PURPOSEDiagnosis of AIDS lymphoma in low-resource settings, like South Africa, is often delayed, leaving patients with limited treatment options. In tuberculosis (TB) endemic regions, overlapping signs and symptoms often lead to diagnostic delays. Assessment of plasma cell-free DNA (cfDNA) by next-generation sequencing (NGS) may expedite the diagnosis of lymphoma but requires high-quality cfDNA.METHODSPeople living with HIV with newly diagnosed aggressive B-cell lymphoma and those with newly diagnosed TB seeking care at Chris Hani Baragwanath Academic Hospital and its surrounding clinics, in Soweto, South Africa, were enrolled in this study. Each participant provided a whole blood specimen collected in cell-stabilizing tubes. Quantity and quality of plasma cfDNA were assessed. NGS of the immunoglobulin heavy chain was performed.RESULTSNine HIV+ patients with untreated lymphoma and eight HIV+ patients with TB, but without lymphoma, were enrolled. All cfDNA quantity and quality metrics were similar between the two groups, except that cfDNA accounted for a larger fraction of recovered plasma DNA in patients with lymphoma. The concentration of cfDNA in plasma also trended higher in patients with lymphoma. NGS of immunoglobulin heavy chain showed robust amplification of DNA, with large amplicons (> 250 bp) being more readily detected in patients with lymphoma. Clonal sequences were detected in five of nine patients with lymphoma, and none of the patients with TB.CONCLUSIONThis proof-of-principle study demonstrates that whole blood collected for cfDNA in a low-resource setting is suitable for sophisticated sequencing analyses, including clonal immunoglobulin NGS. The detection of clonal sequences in more than half of patients with lymphoma shows promise as a diagnostic marker that may be explored in future studies.

Application of a cost-effective DNA extraction protocol for screening transgenic and CRISPR-edited primary goat cells

The genotyping of genetically-modified cells is a crucial step in studies of transgenics and genomic editing with systems such as CRISPR/Cas. The detection of genome editing events can be directly related to the genotyping methodology used, which is influenced by its costs, since many experiments require the analysis of a large number of samples. The aim of this study was to compare the performance of direct lysis methods of genomic DNA (gDNA) extraction for the detection of knockins and knockouts in primary goat cells. Initially, three gDNA extraction protocols (protocol A, heat denaturation/freeze-thaw in water; protocol B, heat denaturation/proteinase K; and protocol C, CellsDirect Kit) were tested using different quantities (1,000, 5,000 and 10,000 cells) and types of goat primary cells (fibroblasts and goat mammary epithelial cells—GMECs) for subsequent validation by PCR amplification of small (GAPDH) and large amplicons (hLF transgene). All protocols were successful in the detection of the small amplicon; however, in GMECs, only protocol B resulted efficient amplification (protocol A—0%, protocol B—93%, protocol C—13.33%, P <0.05). In a proof-of-principle experiment, the TP53 gene was knocked out in GMECs by CRISPR/Cas9-mediated deletion while constructs containing the anti-VEGF monoclonal antibody (pBC-anti-VEGF) and bacterial L-Asparaginase (pBC-ASNase) transgenes were knocked-in separately in fibroblasts. Detection of successful editing was performed using protocol B and PCR. The integration rates of the pBC-ASNase and pBC-anti-VEGF transgenes were 93.6% and 72%, respectively, as per PCR. The efficiency of biallelic editing in GMECs using CRISPR/Cas9 for the TP53 deletion was 5.4%. Our results suggest that protocol B (heat denaturation/proteinase K) can be used as an inexpensive and quick methodology for detecting genetic modifications in different types of primary goat cells, with efficiency rates consistent with values previously described in the literature when using extraction kits or more complex proteinase K formulations.

Abstract 2109: Amplicon design algorithm for single cell targeted DNA sequencing using machine learning

Abstract Background: To realize the promise of precision medicine for cancer, assessing genetic variation present in rare cells and understanding the role that these cells play in the evolution of tumor progression is essential. High throughput single cell DNA targeted sequencing enables detection of rare mutations in cells and identification of subclones defined by co-occurrence of mutations. The big challenge with multiplex sequencing at single cell level is the non-uniform amplification of the targeted regions during PCR. This results in inadequate coverage of mutations of interest in the panel and hence makes genotyping challenging. To address this challenge, we developed a machine learning engine to optimize amplicon design for uniform amplification by making reliable performance prediction. Methods: Multiple panels with various sizes were designed with amplicons spanning a wide range of design properties such as amplicon GC, length, secondary structure prediction, primer specificity. These panels were synthesized and processed through Tapestri single cell DNA platform. The tested amplicons are classified into low-performer, OK-performer and high flyer based on their normalized reads-per-cell value. Design properties and property distribution of the amplicons and the panel are the features. We used random forest classifier to calculate feature importance and analyzed the range of the top features for each class and their significance of variance between classes. These ranges were then used as parameters in the assay design pipeline. Next, we train machine learning models with performance data to develop a performance prediction engine. Results: To test the performance of the design pipeline with new parameters, we designed a small (31), medium (128) and large (287) amplicon panel. Multiple runs were conducted for each panel with different cell types. We were able to achieve high panel performance of 97%, 92% and 88% across the three panels. The new parameters resulted in ~10-20% improvement in panel uniformity. We are working on further optimizing the performance prediction engine by using different ML classification models with K-fold cross validation, training using larger group of amplicons and optimizing features using combinations of properties. Citation Format: Shu Wang, Saurabh Gulati, Dong Kim, Sombeet Sahu, Saurabh Parikh, Nianzhen Li, Manimozhi Manivannan, Nigel Beard. Amplicon design algorithm for single cell targeted DNA sequencing using machine learning [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2109.

Assembly of the threespine stickleback Y chromosome reveals convergent signatures of sex chromosome evolution

BackgroundHeteromorphic sex chromosomes have evolved repeatedly across diverse species. Suppression of recombination between X and Y chromosomes leads to degeneration of the Y chromosome. The progression of degeneration is not well understood, as complete sequence assemblies of heteromorphic Y chromosomes have only been generated across a handful of taxa with highly degenerate sex chromosomes. Here, we describe the assembly of the threespine stickleback (Gasterosteus aculeatus) Y chromosome, which is less than 26 million years old and at an intermediate stage of degeneration. Our previous work identified that the non-recombining region between the X and the Y spans approximately 17.5 Mb on the X chromosome.ResultsWe combine long-read sequencing with a Hi-C-based proximity guided assembly to generate a 15.87 Mb assembly of the Y chromosome. Our assembly is concordant with cytogenetic maps and Sanger sequences of over 90 Y chromosome BAC clones. We find three evolutionary strata on the Y chromosome, consistent with the three inversions identified by our previous cytogenetic analyses. The threespine stickleback Y shows convergence with more degenerate sex chromosomes in the retention of haploinsufficient genes and the accumulation of genes with testis-biased expression, many of which are recent duplicates. However, we find no evidence for large amplicons identified in other sex chromosome systems. We also report an excellent candidate for the master sex-determination gene: a translocated copy of Amh (Amhy).ConclusionsTogether, our work shows that the evolutionary forces shaping sex chromosomes can cause relatively rapid changes in the overall genetic architecture of Y chromosomes.

Isolation and Molecular Characterization of Candida SPP from Poultry with Symptoms of Candidiasis in Ado Ekiti, Nigeria

Aim: The aim of this study is to isolate, identify and characterize Candida spp from cloacal swabs of poultry or birds in Ekiti State University poultry farm, Ago-aduloju poultry farm and Federal Polytechnic of Ado Ekiti poultry farm using molecular method.&#x0D; Place and Period of Study: The study was carried out in the Department of Microbiology, Faculty of Science, Ekiti State University, Ado Ekiti, Nigeria in August 2016.&#x0D; Methodology: Fifty samples of poultry droppings were collected from three farms within Ado Ekiti. The samples were inoculated on Sabourand dextrose agar amended with chloramphenicol. All the fungal isolates were isolated using pour plate method. The isolates were identified based on their morphological, cultural characteristics and molecular analysis.&#x0D; Results: Eight isolates were obtained from a total of fifty samples. Four isolates were identified as Candida albicans strain E10-15 while the fifth isolates was Candida zemplinina strain MCR9. The result showed that three of the eight isolates had small amplicon which were not enough to give the sequence identity of the isolates while the remaining five isolates had large amplicon.&#x0D; Conclusion: The result of the work demonstrated that poultry birds harbor Candida albicans which is a potential pathogenic yeast. This study signifies the need to discover more environmental niches for yeast especially of Candida species and also recommends that poultry birds should always be treated with proper antibiotics to avoid candidiasis.