Biology subjects

Drmanac, R.

Publications and source records attributed to Drmanac, R..

13 recordsLinked to original sources

bioRxiv2025

Capture-C MPRA: A high-throughput method to simultaneously characterize promoter interactions and regulatory activity

Cis regulatory elements (CREs) interact with their target promoters over long genomic distances and can be identified using chromatin conformation capture (3C) assays. Their regulatory activity can be functionally characterized in a high-throughput manner using massively parallel reporter assays (MPRAs) that generally test an enhancer alongside a minimal promoter. Here, we developed a novel technology called Capture-C MPRA (ccMPRA) that combines both technologies and can simultaneously obtain chromatin interactions and measure CRE activity alongside their target promoters. We utilized ccMPRA to analyze the regulatory activity of 650 promoters interacting with 42,719 sequences. As C-based techniques also capture isolated promoters, we were able to obtain promoter baseline activity, enabling the identification of both enhancers and silencers. Analysis of CREs interacting with more than one promoter showed significant activity differences depending on the promoter. In summary, ccMPRA can simultaneously characterize chromatin interactions and regulatory activity, allowing to further dissect regulatory grammar.

genomics↗

bioRxiv2023

Assembly and analysis of the genome of Notholithocarpus densiflorus

Tanoak (Notholithocarpus densiflorus) is an evergreen tree in the Fagaceae family found in California and southern Oregon. Historically, tanoak acorns were an important food source for Native American tribes and the bark was used extensively in the leather tanning process. Long considered a disjunct relictual element of the Asian stone oaks (Lithocarpus spp.), phylogenetic analysis has determined that the tanoak is an example of convergent evolution. Tanoaks are deeply divergent from oaks (Quercus) of the Pacific Northwest and comprise a new genus with a single species. These trees are highly susceptible to sudden oak death (SOD), a plant pathogen (Phytophthora ramorum) that has caused widespread mortality of tanoaks. Here, we set out to assemble the genome and perform comparative studies among a number of individuals that demonstrated varying levels of susceptibility to SOD. First, we sequenced and de novo assembled a draft reference genome of N. densiflorus using co-barcoded library processing methods and an MGI DNBSEQ-G400 sequencer. To increase the contiguity of the final assembly, we also sequenced Oxford Nanopore (ONT) long reads to 30X coverage. To our knowledge, the draft genome reported here is one of the more contiguous and complete genomes of a tree species published until now, with a contig N50 of [~]1.2 Mb and a scaffold N50 of [~]2.1 Mb. In addition, we sequenced 11 genetically distinct individuals and mapped these onto the draft reference genome enabling the discovery of almost 25 million single nucleotide polymorphisms and [~]4.4 million small insertions and deletions. Finally, using co-barcoded data we were able to generate complete haplotype coverage of all 11 genomes.

genomics↗

bioRxiv2021

High-resolution single-molecule long-fragment rRNA gene amplicon sequencing for uncultured bacterial and fungal communities

Although several large-scale environmental microbial projects have been initiated in the past two decades, understanding of the role of complex microbiotas is still constrained by problems of detecting and identifying unknown microorganisms1-6.Currently, hypervariable regions of rRNA genes as well as internal transcribed spacer regions are broadly used to identify bacteria and fungi within complex communities7,8, but taxonomic and phylogenetic resolution is hampered by insufficient sequencing length9-11. Direct sequencing of full length rRNA genes is currently limited by read length using second generation sequencing or sacrificed quality and throughput by using single molecule sequencing. We developed a novel method to sequence and assemble nearly full length rRNA genes using second generation sequencing.Benchmarking was performed on mock bacterial and fungal communities as well as two forest soil samples. The majority of rRNA gene sequences of all species in the mock community samples were successfully recovered with identities above 99.5% compared to the reference sequences. For soil samples we obtained exquisite coverage with identification of a large number of putative new species, as well as high abundance correlation between replicates. This approach provides a cost-effective method for obtaining extensive and accurate information on complex environmental microbial communities.

microbiology↗

bioRxiv2020

Regulation of specific cell clusters in TCR-T cells responding to differential expression of tumor PD-L1

PD-L1 signaling is important in regulating T cell function and keeping balance of tumor microenvironment, but its role in modifying TCR-T cell cytotoxicity remains unknown. MART-1-specific TCR-T cells (TCR-TMART-1) were stimulated by MEL-526 tumor cells expressing different proportions of PD-L1 and used to perform cytotoxicity assays and single-cell RNA sequencing. Percentage changes of different specific cell clusters were analyzed. The percentage of cluster HLA-DR+CD38+CD8+ was upregulated after antigen stimulation and tumor PD-L1 modified TCR-T cell function through downregulating the percentages of clusters HLA-DR+CD28+CD8+ and HLA-DR+CD38+CD8+ which were higher in TCR-TMART-1 than in Tnull.

immunology↗

bioRxiv2020

Reusable and Sensitive Detection Method for Exonuclease III Activity by DNB Nanoarrays Based on cPAS Sequencing Technology

AbstractionIn this article, we have designed a sensitive and recycled DNB (DNA nanoball) nanoarrays sequencing complex structures based on BGISEQ-500RS sequencer for the monitoring performance of Exo III activity. In the shortage of Exo III, the effective number ratio of DNB would be captured by an optical system due to one fluorescent. In contrast, in the presence of Exo III, some DNB would disappear or discard from the fields of the optical system by fluorescence extinction and uncleaned fluorescent, respectively. As a result, the effective number of DNB of this strategy was relative to the concentration of Exo III. For Exo III, our strategy showed a highly sensitive linear response in the low detection range of 0.01 U/mL to 0.5 U/mL, with detection limits below 0.01 U/mL. With the comparison between DNB nanoarrays and other fluorescent sensors, this study possessed superior sensitivity, selectivity, and reusability, accompanying with the low cost and simple setup.

biochemistry↗

bioRxiv2020

Comparison of long read methods for sequencing and assembly of a plant genome

Sequencing technologies have advanced to the point where it is possible to generate high accuracy, haplotype resolved, chromosome scale assemblies. Several long read sequencing technologies are available on the market and a growing number of algorithms have been developed over the last years to assemble the reads generated by those technologies. When starting a new genome project, it is therefore challenging to select the most cost-effective sequencing technology as well as the most appropriate software for assembly and polishing. For this reason, it is important to benchmark different approaches applied to the same sample. Here, we report a comparison of three long read sequencing technologies applied to the de novo assembly of a plant genome, Macadamia jansenii. We have generated sequencing data using Pacific Biosciences (Sequel I), Oxford Nanopore Technologies (PromethION) and BGI (single-tube Long Fragment Read) technologies for the same sample. Several assemblers were benchmarked in the assembly of PacBio and Nanopore reads. Results obtained from combining long read technologies or short read and long read technologies are also presented. The assemblies were compared for contiguity, accuracy and completeness as well as sequencing costs and DNA material requirements. Overall, the three long read technologies produced highly contiguous and complete genome assemblies of Macadamia jansenii. At the time of sequencing, the cost associated with each method was significantly different but continuous improvements in technologies have resulted in greater accuracy, increased throughput and reduced costs. We propose updating this comparison regularly with reports on significant iterations of the sequencing technologies.

genomics↗

bioRxiv2020

CoolMPS™: Advanced massively parallel sequencing using antibodies specific to each natural nucleobase

Massively parallel sequencing (MPS) on DNA nanoarrays provides billions of reads at relatively low cost and enables a multitude of genomic applications. Further improvement in read length, sequence quality and cost reduction will enable more affordable and accurate comprehensive health monitoring tests. Currently the most efficient MPS uses dye-labeled reversibly terminated nucleotides (RTs) that are expensive to make and challenging to incorporate. Furthermore, a part of the dye-linker (scar) remains on the nucleobase after cleavage and interferes with subsequent sequencing cycles. We describe here the development of a novel MPS chemistry (CoolMPS) utilizing unlabeled RTs and four natural nucleobase-specific fluorescently labeled antibodies with fast (30 sec) binding. We implemented CoolMPS on MGIs PCR-free DNBSEQ MPS platform using arrays of 200nm DNA nanoballs (DNBs) generated by rolling circle replication and demonstrate 3-fold improvement in signal intensity and elimination of scar interference. Single-end 100-400 base and pair-end 2x150 base reads with high quality were readily generated with low out-of-phase incorporation. Furthermore, DNBs with less than 50 template copies were successfully sequenced by strong-signal CoolMPS with 3-times higher accuracy than in standard MPS. CoolMPS chemistry based on natural nucleobases has potential to provide longer, more accurate and less expensive MPS reads, including highly accurate "4-color sequencing" on the most efficient dye-crosstalk-free 2-color imagers with an estimated sequencing error rate of 0.00058% (one error in 170,000 base calls) in a proof-of-concept demonstration.

genomics↗

bioRxiv2019

Advanced Whole Genome Sequencing Using a Complete PCR-free Massively Parallel Sequencing (MPS) Workflow

BackgroundSystematic errors can be introduced from DNA amplification during massively parallel sequencing (MPS) library preparation and sequencing array formation. Polymerase chain reaction (PCR)-free genomic library preparation methods were previously shown to improve whole genome sequencing (WGS) quality on the Illumina platform, especially in calling insertions and deletions (InDels). We hypothesized that substantial InDel errors continue to be introduced by the remaining PCR step of DNA cluster generation. In addition to library preparation and sequencing, data analysis methods are also important for the accuracy of the output data.In recent years, several machine learning variant calling pipelines have emerged, which can correct the systematic errors from MPS and improve the data performance of variant calling. ResultsHere, PCR-free libraries were sequenced on the PCR-free DNBSEQ arrays from MGI Tech Co., Ltd. (referred to as MGI) to accomplish the first true PCR-free WGS which the whole process is truly not only PCR-free during library preparation but also PCR-free during sequencing. We demonstrated that PCR-based WGS libraries have significantly (about 5 times) more InDel errors than PCR-free libraries.Furthermore, PCR-free WGS libraries sequenced on the PCR-free DNBSEQ platform have up to 55% less InDel errors compared to the NovaSeq platform, confirming that DNA clusters contain PCR-generated errors.In addition, low coverage bias and less than 1% read duplication rate was reproducibly obtained in DNBSEQ PCR-free using either ultrasonic or enzymatic DNA fragmentation MGI kits combined with MGISEQ-2000. Meanwhile, variant calling performance (single-nucleotide polymorphisms (SNPs) F-score>99.94%, InDels F-score>99.6%) exceeded widely accepted standards using machine learning (ML) methods (DeepVariant or DNAscope). ConclusionsEnabled by the new PCR-free library preparation kits, ultra high-thoughput PCR-free sequencers and ML-based variant calling, true PCR-free DNBSEQ WGS provides a powerful solution for improving WGS accuracy while reducing cost and analysis time, thus facilitating future precision medicine, cohort studies, and large population genome projects.

genomics↗

bioRxiv2018

3’ Branch Ligation: A Novel Method to Ligate Non-Complementary DNA to Recessed or Internal 3’OH Ends in DNA or RNA

Nucleic acid ligases are crucial enzymes that repair breaks in DNA or RNA during synthesis, repair and recombination. Various molecular tools have been developed using the diverse activities of DNA/RNA ligases. Herein, we demonstrate a non-conventional ability of T4 DNA ligase to join 5 phosphorylated blunt-end double-stranded DNA to DNA breaks at 3 recessive ends, gaps, or nicks to form a 3 branch structure. Therefore, this base pairing-independent ligation is termed 3 branch ligation (3BL). In an extensive study of optimal ligation conditions, similar to blunt-end ligation, the presence of 10% PEG-8000 in the ligation buffer significantly increased ligation efficiency. A low level of nucleotide preference was observed at the junction sites using different synthetic DNAs. Furthermore, we discovered that T4 DNA ligase efficiently ligated DNA to the 3 recessed end of RNA, not to that of DNA, in a DNA/RNA hybrid, whereas RNA ligases are less efficient in this reaction. These novel properties of T4 DNA ligase can be utilized as a broad molecular technique in many important applications. We performed a proof-of-concept study of a new directional tagmentation protocol for next generation sequencing (NGS) library construction that eliminates inverted adapters and allows sample barcode insertion adjacent to genomic DNA. 3BL after single transposon tagmentation can theoretically achieve 100% usable template, and our empirical data demonstrate that the new approach produced higher yield compared with traditional double transposon or Y transposon tagmentation. We further explore the potential use of 3BL for preparing targeted RNA NGS libraries with mitigated structure-based bias and adapter dimer problems.

molecular biology↗

bioRxiv2018

Reliable Multiplex Sequencing with Rare Index Mis-Assignment on DNB-Based NGS Platform

BackgroundMassively-parallel-sequencing, coupled with sample multiplexing, has made genetic tests broadly affordable. However, intractable index mis-assignments (commonly exceeds 1%) were repeatedly reported on some widely used sequencing platforms.\n\nResultsHere, we investigated this quality issue on BGI sequencers using three library preparation methods: whole genome sequencing (WGS) with PCR, PCR-free WGS, and two-step targeted PCR. BGIs sequencers utilize a unique DNB technology which uses rolling circle replication for DNA-nanoball preparation; this linear amplification is PCR free and can avoid error accumulation. We demonstrated that single index mis-assignment from free indexed oligos occurs at a rate of one in 36 million reads, suggesting virtually no index hopping during DNB creation and arraying. Furthermore, the DNB-based NGS libraries have achieved an unprecedentedly low sample-to-sample mis-assignment rate of 0.0001% to 0.0004% under recommended procedures.\n\nConclusionsSingle indexing with DNB technology provides a simple but effective method for sensitive genetic assays with large sample numbers.

genomics↗

bioRxiv2018

Single tube bead-based DNA co-barcoding for cost effective and accurate sequencing, haplotyping, and assembly

Obtaining accurate sequences from long DNA molecules is very important for genome assembly and other applications. Here we describe single tube long fragment read (stLFR), a technology that enables this a low cost. It is based on adding the same barcode sequence to sub-fragments of the original long DNA molecule (DNA co-barcoding). To achieve this efficiently, stLFR uses the surface of microbeads to create millions of miniaturized barcoding reactions in a single tube. Using a combinatorial process up to 3.6 billion unique barcode sequences were generated on beads, enabling practically non-redundant co-barcoding with 50 million barcodes per sample. Using stLFR, we demonstrate efficient unique co-barcoding of over 8 million 20-300 kb genomic DNA fragments. Analysis of the genome of the human genome NA12878 with stLFR demonstrated high quality variant calling and phasing into contigs up to N50 34 Mb. We also demonstrate detection of complex structural variants and complete diploid de novo assembly of NA12878. These analyses were all performed using single stLFR libraries and their construction did not significantly add to the time or cost of whole genome sequencing (WGS) library preparation. stLFR represents an easily automatable solution that enables high quality sequencing, phasing, SV detection, scaffolding, cost-effective diploid de novo genome assembly, and other long DNA sequencing applications.

genomics↗

bioRxiv2017

Significant abundance of cis configurations of mutations in diploid human genomes

To fully understand human genetic variation, one must assess the specific distribution of variants between the two chromosomal homologues of genes, and any functional units of interest, as the phase of variants can significantly impact gene function and phenotype. To this end, we have systematically analyzed 18,121 autosomal protein-coding genes in 1,092 statistically phased genomes from the 1000 Genomes Project, and an unprecedented number of 184 experimentally phased genomes from the Personal Genome Project. Here we show that mutations predicted to functionally alter the protein, and coding variants as a whole, are not randomly distributed between the two homologues of a gene, but do occur significantly more frequently in cis-than trans-configurations, with cis/trans ratios of [~]60:40. Significant cis-abundance was observed in virtually all individual genomes in all populations. Nearly all variable genes exhibited either cis, or trans configurations of protein-altering mutations in significant excess, allowing distinction of cis- and trans-abundant genes. These common patterns of phase were largely constituted by a shared, global set of phase-sensitive genes. We show significant enrichment of this global set with gene sets indicating its involvement in adaptation and evolution. Moreover, cis- and trans-abundant genes were found functionally distinguishable, and exhibited strikingly different distributional patterns of protein-altering mutations. This work establishes common patterns of phase as key characteristics of diploid human exomes and provides evidence for their potential functional significance. Thus, it highlights the importance of phase for the interpretation of protein-coding genetic variation, challenging the current conceptual and functional interpretation of autosomal genes.

genomics↗

bioRxiv2017

Advanced whole genome sequencing and analysis of fetal genomes from amniotic fluid

Amniocentesis is typically performed to identify large chromosomal abnormalities within the fetus. Here we demonstrate that it is feasible to generate an accurate whole genome sequence (WGS) of a fetus from an amniotic sample. DNA from cells and the amniotic fluid were isolated and sequenced from 31 amniocenteses. Concordance of variant calls between the two DNA sources and with parental libraries was high. Two fetal genomes were found to harbor potentially detrimental variants in CHD8 and LRP1, variations in these genes have been associated with Autism Spectrum Disorder (ASD) and Keratosis pilaris atrophicans, respectively. We also discovered drug sensitivities and carrier information of fetuses for a variety of diseases. In this study, we demonstrate for the first time the sequencing of the whole genome of fetuses from amniotic fluid and show that much more information than large chromosomal abnormalities can be gained from an amniocentesis.

genomics↗