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Microbial genomes produced by standard single-cell amplification methods are largely incomplete. Here, we show that primary template-directed amplification (PTA), a novel single-cell amplification technique, generated nearly complete genomes from three bacterial isolate species. Furthermore, taxonomically diverse genomes recovered from aquatic and soil microbiomes using PTA had a median completeness of 81%, whereas genomes from standard multiple displacement amplification-based approaches were usually

Abstract Background Identification and characterization of novel, faithful and processive DNA polymerases is a driving force in the development of DNA amplification methods. Purification of proteins from natural phages is often time-consuming, cumbersome and low yielding. Escherichia coli is a host bacterium widely used for the production of recombinant proteins, is the cell factory of choice for in vitro studies of phage protein function. Results We expressed the gene encoding Enterococcus faecium phage IME199 DNA polymerase (IME199 DNAP) in Escherichia coli BL21(DE3), and characterized protein function. IME199 DNAP has 3′-5′ exonuclease activity, but does not have 5′-3′ exonuclease activity. In addition, IME199 DNAP has dNTP-dependent 5′-3′ polymerase activity and can amplify DNA at 15–35 °C and a pH range of 5.5–9.5. The amino acid residues Asp30, Glu32, Asp112 and Asp251 are the 3′-5′ exonuclease active sites of IME199 DNAP, while residues Asp596 and Tyr639 are essential for DNA synthesis by IME199 DNAP. More importantly, the IME199 DNAP has strand displacement and processive synthesis capabilities, and can perform rolling circle amplification and multiple displacement amplification with very low error rates (approximately 3.67 × 10–6). Conclusions A novel family B DNA polymerase was successfully overproduced in Escherichia coli BL21(DE3). Based on the characterized properties, IME199 DNAP is expected to be developed as a high-fidelity polymerase for DNA amplification at room temperature.

Background: Identification and characterization of novel, faithful and processive DNA polymerases is a driving force in the development of DNA amplification methods. Purification of proteins from natural phages is often time-consuming, cumbersome and low yielding. Escherichia coli is a host bacterium widely used for the production of recombinant proteins, is the cell factory of choice for in vitro studies of phage protein function. Results: We expressed the gene encoding Enterococcus faecium phage IME199 DNA polymerase (IME199 DNAP) in Escherichia coli BL21(DE3), and characterized protein function. IME199 DNAP has 3′-5′ exonuclease activity, but does not have 5′-3′ exonuclease activity. In addition, IME199 DNAP has dNTP-dependent 5′-3′ polymerase activity and can amplify DNA at 15–35 °C and a pH range of 5.5–9.5. The amino acid residues Asp30, Glu32, Asp112 and Asp251 are the 3′-5′ exonuclease active sites of IME199 DNAP, while residues Asp596 and Tyr639 are essential for DNA synthesis by IME199 DNAP. More importantly, the IME199 DNAP has strand displacement and processive synthesis capabilities, and can perform rolling circle amplification and multiple displacement amplification with very low error rates (approximately 3.67 × 10–6). Conclusions: A novel family B DNA polymerase was successfully overproduced in Escherichia coli BL21(DE3). Based on the characterized properties, IME199 DNAP is expected to be developed as a high-fidelity polymerase for DNA amplification at room temperature. [ABSTRACT FROM AUTHOR]

Motivation Multiple displacement amplification (MDA) has become the most commonly used method of whole genome amplification, generating a vast amount of DNA with higher molecular weight and greater genome coverage. Coupling with long-read sequencing, it is possible to sequence the amplicons of over 20 kb in length. However, the formation of chimeric sequences (chimeras, expressed as structural errors in sequencing data) in MDA seriously interferes with the bioinformatics analysis but its influence on long-read sequencing data is unknown. Results We sequenced the phi29 DNA polymerase-mediated MDA amplicons on the PacBio platform and analyzed chimeras within the generated data. The 3rd-ChimeraMiner has been constructed as a pipeline for recognizing and restoring chimeras into the original structures in long-read sequencing data, improving the efficiency of using TGS data. Five long-read datasets and one high-fidelity long-read dataset with various amplification folds were analyzed. The result reveals that the mis-priming events in amplification are more frequently occurring than widely perceived, and the propor tion gradually accumulates from 42% to over 78% as the amplification continues. In total, 99.92% of recognized chimeric sequences were demonstrated to be artifacts, whose structures were wrongly formed in MDA instead of existing in original genomes. By restoring chimeras to their original structures, the vast majority of supplementary alignments that introduce false-positive structural variants are recycled, removing 97% of inversions on average and contributing to the analysis of structural variation in MDA-amplified samples. The impact of chimeras in long-read sequencing data analysis should be emphasized, and the 3rd-ChimeraMiner can help to quantify and reduce the influence of chimeras. Availability and implementation The 3rd-ChimeraMiner is available on GitHub, https://github.com/dulunar/3rdChimeraMiner. [ABSTRACT FROM AUTHOR]

Multiple displacement amplification (MDA) based on isothermal random priming and high fidelity phi29 DNA polymerase-mediated processive extension has revolutionized the field of whole genome amplification by enabling the amplification of minute amounts of DNA, such as from a single cell, generating vast amounts of DNA with high genome coverage. Despite its advantages, MDA has its own challenges, one of the grandest being the formation of chimeric sequences (chimeras), which presents in all MDA products and seriously disturbs the downstream analysis. In this review, we provide a comprehensive overview of current research on MDA chimeras. We first reviewed the mechanisms of chimera formation and chimera detection methods. We then systematically summarized the characteristics of chimeras, including overlap, chimeric distance, chimeric density, and chimeric rate, as found in independently published sequencing data. Finally, we reviewed the methods used to process chimeric sequences and their impacts on the improvement of data utilization efficiency. The information presented in this review will be useful for those interested in understanding the challenges with MDA and in improving its performance.

In the same way that specialized DNA polymerases (DNAPs) replicate cellular and viral genomes, only a handful of dedicated proteins from various natural origins as well as engineered versions are appropriate for competent exponential amplification of whole genomes and metagenomes (WGA). Different applications have led to the development of diverse protocols, based on various DNAPs. Isothermal WGA is currently widely used due to the high performance of Φ29 DNA polymerase, but PCR-based methods are also available and can provide competent amplification of certain samples. Replication fidelity and processivity must be considered when selecting a suitable enzyme for WGA. However, other properties, such as thermostability, capacity to couple replication, and double helix unwinding, or the ability to maintain DNA replication opposite to damaged bases, are also very relevant for some applications. In this review, we provide an overview of the different properties of DNAPs widely used in WGA and discuss their limitations and future research directions. [ABSTRACT FROM AUTHOR]

Marine invertebrate microbiomes have been a rich source of bioactive compounds and interesting genomic features. In cases where the achievable amounts of metagenomic DNA are too low for direct sequencing, multiple displacement amplification (MDA) can be used for whole genome amplification. However, MDA has known limitations which can affect the quality of the resulting genomes and metagenomes. In this study, we evaluated the conservation of biosynthetic gene clusters (BGCs) and enzymes in MDA products from low numbers of prokaryotic cells (estimated 2–850). Marine invertebrate microbiomes collected from Arctic and sub-Arctic areas served as source material. The cells were separated from the host tissue, lysed, and directly subjected to MDA. The MDA products were sequenced by Illumina sequencing. Corresponding numbers of bacteria from a set of three reference strains were treated the same way. The study demonstrated that useful information on taxonomic, BGC, and enzyme diversities was obtainable from such marginal quantities of metagenomic material. Although high levels of assembly fragmentation resulted in most BGCs being incomplete, we conclude that this genome mining approach has the potential to reveal interesting BGCs and genes from hard-to-reach biological sources. [ABSTRACT FROM AUTHOR]

Background: Viruses play critical roles in the marine environment because of their interactions with an extremely broad range of potential hosts. Many studies of viruses in seawater have been published, but viruses that inhabit marine animals have been largely neglected. Oysters are keystone species in coastal ecosystems, yet as filter-feeding bivalves with very large roosting numbers and species co-habitation, it is not clear what role they play in marine virus transmission and coastal microbiome regulation. Results: Here, we report a Dataset of Oyster Virome (DOV) that contains 728,784 nonredundant viral operational taxonomic unit contigs (≥ 800 bp) and 3473 high-quality viral genomes, enabling the first comprehensive overview of both DNA and RNA viral communities in the oyster Crassostrea hongkongensis. We discovered tremendous diversity among novel viruses that inhabit this oyster using multiple approaches, including reads recruitment, viral operational taxonomic units, and high-quality virus genomes. Our results show that these viruses are very different from viruses in the oceans or other habitats. In particular, the high diversity of novel circoviruses that we found in the oysters indicates that oysters may be potential hotspots for circoviruses. Notably, the viruses that were enriched in oysters are not random but are well-organized communities that can respond to changes in the health state of the host and the external environment at both compositional and functional levels. Conclusions: In this study, we generated a first "knowledge landscape" of the oyster virome, which has increased the number of known oyster-related viruses by tens of thousands. Our results suggest that oysters provide a unique habitat that is different from that of seawater, and highlight the importance of filter-feeding bivalves for marine virus exploration as well as their essential but still invisible roles in regulating marine ecosystems. APUP9ceFw5rgMggB2pWS5V Video Abstract [ABSTRACT FROM AUTHOR]

Abstract Background In preimplantation genetic testing for aneuploidy (PGT-A), appropriate evaluation of mosaic embryos is important because of the adverse implications of transferring embryos with high-level mosaicism or discarding those with low-level mosaicism. Despite the availability of multiple reliable techniques for PGT-A, data comparing the detection of mosaicism using these techniques are scarce. To address this gap in the literature, we compared the detection ability of the two most commonly used PGT-A platforms, next-generation sequencing (NGS) and the single-nucleotide polymorphism (SNP) array, for mosaic embryos. Results We retrospectively reviewed the data of PGT-A or preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR) conducted at our center from January 2018 to October 2020, and selected blastocysts that underwent aneuploidy screening with both an SNP array and NGS. Trophectoderm biopsy, multiple displacement amplification (MDA), and aneuploidy screening with an SNP array were conducted on the enrolled blastocysts. When the SNP array indicated mosaicism, NGS was performed on the corresponding MDA product for verification. Among the 105 blastocysts diagnosed with mosaicism with the SNP array, 80 (76.19%) showed mosaicism in NGS, with complete and partial concordance rates of 47.62% (50/105) and 18.10% (19/105), respectively. The complete discordance rate of the two platforms was 34.29% (36/105). That is, 10.48% (11/105) of the blastocysts were diagnosed with completely different types of mosaicism with the two platforms, while 13.33% (14/105) and 10.48% (11/105) of the embryos diagnosed as showing mosaicism with SNP were detected as showing aneuploidy and euploidy with NGS, respectively. Conclusions The consistency of NGS and the SNP array in the diagnosis of embryo mosaicism is extremely low, indicating the need for larger and well-designed studies to determine which platform is more accurate in detecting mosaic embryos.

Sequencing of single bacterial and archaeal cells is an important methodology that provides access to the genetic makeup of uncultivated microorganisms. We here describe the high-throughput fluorescence-activated cell sorting-based isolation of single cells from the environment, their lysis and strand displacement-mediated whole genome amplification. We further outline 16S rRNA gene sequence-based screening of single-cell amplification products, their preparation for Illumina sequencing libraries, and finally propose computational methods for read and contig level quality control of the resulting sequence data.

ABSTRACT Metagenomic next-generation sequencing (mNGS) offers a hypothesis-free approach for pathogen detection, but its applicability in clinical diagnosis, in addition to other factors, remains limited due to complicated library construction. The present study describes a PCR-free isothermal workflow for mNGS targeting RNA, based on a multiple displacement amplification, termed circular whole-transcriptome amplification (cWTA), as the template is circularized before amplification. The cWTA approach was validated with clinical samples and nanopore sequencing. Reads homologous to dengue virus 2 and chikungunya virus were detected in clinical samples from Bangladesh and Brazil, respectively. In addition, the practicality of a high-throughput detection system that combines mNGS and a group testing algorithm termed mNGS screening enhanced by a group testing algorithm (mEGA) was established. This approach enabled significant library size reduction while permitting trackability between samples and diagnostic results. Serum samples of patients with undifferentiated febrile illnesses from Vietnam (n = 43) were also amplified with cWTA, divided into 11 pools, processed for library construction, and sequenced. Dengue virus 2, hepatitis B virus, and parvovirus B19 were successfully detected without prior knowledge of their existence. Collectively, cWTA with the nanopore platform opens the possibility of hypothesis-free on-site comprehensive pathogen diagnosis, while mEGA contributes to the scaling up of sample throughput. IMPORTANCE Given the breadth of pathogens that cause infections, a single approach that can detect a wide range of pathogens is ideal but is impractical due to the available tests being highly specific to a certain pathogen. Recent developments in sequencing technology have introduced mNGS as an alternative that provides detection of a wide-range of pathogens by detecting the presence of their nucleic acids in the sample. However, sequencing library preparation is still a bottleneck, as it is complicated, costly, and time-consuming. In our studies, alternative approaches to optimize library construction for mNGS were developed. This included isothermal nucleic acid amplification and expansion of sample throughput with a group testing algorithm. These methods can improve the utilization of mNGS as a diagnostic tool and can serve as a high-throughput screening system aiding infectious disease surveillance.

In the same way that specialized DNA polymerases (DNAPs) replicate cellular and viral genomes, only a handful of dedicated proteins from various natural origins as well as engineered versions are appropriate for competent exponential amplification of whole genomes and metagenomes (WGA). Different applications have led to the development of diverse protocols, based on various DNAPs. Isothermal WGA is currently widely used due to the high performance of Φ29 DNA polymerase, but PCR-based methods are also available and can provide competent amplification of certain samples. Replication fidelity and processivity must be considered when selecting a suitable enzyme for WGA. However, other properties, such as thermostability, capacity to couple replication, and double helix unwinding, or the ability to maintain DNA replication opposite to damaged bases, are also very relevant for some applications. In this review, we provide an overview of the different properties of DNAPs widely used in WGA and discuss their limitations and future research directions.

Purpose: To determine the application value of next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidies (PGT-A). Methods: We conducted a retrospective case–control study on a cohort of frozen-thawed embryo transfer (FET) cycles following preimplantation genetic testing for monogenic disorders (PGT-M) between 2014 and 2017. Cycles that produced live births or early miscarriages were divided into live birth group (n = 76) or miscarriage group (n = 19), respectively. The NGS-based aneuploidy screening was performed on the multiple displacement amplification (MDA) products of the embryonic trophectoderm biopsy samples that were cryopreserved following PGT-M. Results: In the live birth group, 75% (57/76) embryos were euploid and 14.5% (11/76) were aneuploid. The remaining 10.5% (8/76) embryos were NGS-classified mosaic with the high- (≥ 50%) and low-level (< 50%) mosaicism rates at 7.9% (6/76) and 2.6% (2/76), respectively. In the miscarriage group, only 23.5% (4/17) embryos were aneuploid, while 58.8% (10/17) were euploid and 17.6% (3/17) were NGS-classified mosaic with the high- and low-level mosaicism rates at 11.8% (2/17) and 5.9% (1/17), respectively. For live birth and miscarriage groups, the transferable rate was 82.9% (63/76) and 70.6% (12/17), respectively, whereas the untransferable rate was 17.1% (13/76) and 29.4% (5/17), respectively. Conclusion: The application of NGS-based PGT-A remains questionable, as it may cause at least one in six embryos with reproductive potential to be discarded and prevent miscarriage in less than one in three embryos in single-gene disease carriers. [ABSTRACT FROM AUTHOR]

Successful whole genome amplification (WGA) is a cornerstone of contemporary preimplantation genetic testing (PGT). Choosing the most suitable WGA technique for PGT can be particularly challenging because each WGA technique performs differently in combination with different downstream processing and detection methods. The aim of this review is to provide insight into the performance and drawbacks of DOP-PCR, MDA and MALBAC, as well as the hybrid WGA techniques most widely used in PGT. As the field of PGT is moving towards a wide adaptation of comprehensive massively parallel sequencing (MPS)-based approaches, we especially focus our review on MPS parameters and detection opportunities of WGA-amplified material, i.e., mappability of reads, uniformity of coverage and its influence on copy number variation analysis, and genomic coverage and its influence on single nucleotide variation calling. The ability of MDA-based WGA solutions to better cover the targeted genome and the ability of PCR-based solutions to provide better uniformity of coverage are highlighted. While numerous comprehensive PGT solutions exploiting different WGA types and adjusted bioinformatic pipelines to detect copy number and single nucleotide changes are available, the ones exploiting MDA appear more advantageous. The opportunity to fully analyse the targeted genome is influenced by the MPS parameters themselves rather than the solely chosen WGA. [ABSTRACT FROM AUTHOR]

Uniform amplification of low input DNA is important for applications across biology, including single-cell genomics, forensic science, and microbial and viral sequencing. However, the requisite biochemical amplification methods are prone to bias, skewing sequence proportions and obscuring signals relating to copy number. Digital droplet multiple displacement amplification enables uniform amplification, but requires expert knowledge of microfluidics to generate monodisperse emulsions. In addition, existing microfluidic methods are tedious and labor intensive for preparing many samples. Here, we introduce rapid emulsification multiple displacement amplification, a method to generate monodisperse droplets with a hand-held syringe and hierarchical droplet splitter. While conventional microfluidic devices require >10 minutes to emulsify a sample, our system takes tens of seconds and yields data of equivalent quality. We demonstrate the approach by using it to accurately measure copy number variation in single cancer cells.

Marine invertebrate microbiomes have been a rich source of bioactive compounds and interesting genomic features. In cases where the achievable amounts of metagenomic DNA are too low for direct sequencing, multiple displacement amplification (MDA) can be used for whole genome amplification. However, MDA has known limitations which can affect the quality of the resulting genomes and metagenomes. In this study, we evaluated the conservation of biosynthetic gene clusters (BGCs) and enzymes in MDA products from low numbers of prokaryotic cells (estimated 2–850). Marine invertebrate microbiomes collected from Arctic and sub-Arctic areas served as source material. The cells were separated from the host tissue, lysed, and directly subjected to MDA. The MDA products were sequenced by Illumina sequencing. Corresponding numbers of bacteria from a set of three reference strains were treated the same way. The study demonstrated that useful information on taxonomic, BGC, and enzyme diversities was obtainable from such marginal quantities of metagenomic material. Although high levels of assembly fragmentation resulted in most BGCs being incomplete, we conclude that this genome mining approach has the potential to reveal interesting BGCs and genes from hard-to-reach biological sources.

Background: In preimplantation genetic testing for aneuploidy (PGT-A), appropriate evaluation of mosaic embryos is important because of the adverse implications of transferring embryos with high-level mosaicism or discarding those with low-level mosaicism. Despite the availability of multiple reliable techniques for PGT-A, data comparing the detection of mosaicism using these techniques are scarce. To address this gap in the literature, we compared the detection ability of the two most commonly used PGT-A platforms, next-generation sequencing (NGS) and the single-nucleotide polymorphism (SNP) array, for mosaic embryos. Results: We retrospectively reviewed the data of PGT-A or preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR) conducted at our center from January 2018 to October 2020, and selected blastocysts that underwent aneuploidy screening with both an SNP array and NGS. Trophectoderm biopsy, multiple displacement amplification (MDA), and aneuploidy screening with an SNP array were conducted on the enrolled blastocysts. When the SNP array indicated mosaicism, NGS was performed on the corresponding MDA product for verification. Among the 105 blastocysts diagnosed with mosaicism with the SNP array, 80 (76.19%) showed mosaicism in NGS, with complete and partial concordance rates of 47.62% (50/105) and 18.10% (19/105), respectively. The complete discordance rate of the two platforms was 34.29% (36/105). That is, 10.48% (11/105) of the blastocysts were diagnosed with completely different types of mosaicism with the two platforms, while 13.33% (14/105) and 10.48% (11/105) of the embryos diagnosed as showing mosaicism with SNP were detected as showing aneuploidy and euploidy with NGS, respectively. Conclusions: The consistency of NGS and the SNP array in the diagnosis of embryo mosaicism is extremely low, indicating the need for larger and well-designed studies to determine which platform is more accurate in detecting mosaic embryos. [ABSTRACT FROM AUTHOR]

In this study, we addressed different aspects regarding the implementation of quasimetagenomic sequencing as a hybrid surveillance method in combination with enrichment for early detection of Listeria monocytogenes in the food industry. Different experimental enrichment cultures were used, comprising seven L. monocytogenes strains of different sequence types (STs), with and without a background microbiota community. To assess whether the proportions of the different STs changed over time during enrichment, the growth and population dynamics were assessed using dapE colony sequencing and dapE and 16S rRNA amplicon sequencing. There was a tendency of some STs to have a higher relative abundance during the late stage of enrichment when L. monocytogenes was enriched without background microbiota. When coenriched with background microbiota, the population dynamics of the different STs was more consistent over time. To evaluate the earliest possible time point during enrichment that allows the detection of L. monocytogenes and at the same time the generation of genetic information that enables an estimation regarding the strain diversity in a sample, quasimetagenomic sequencing was performed early during enrichment in the presence of the background microbiota using Oxford Nanopore Technologies Flongle and Illumina MiSeq sequencing. The application of multiple displacement amplification (MDA) enabled detection of L. monocytogenes (and the background microbiota) after only 4 h of enrichment using both applied sequencing approaches. The MiSeq sequencing data additionally enabled the prediction of cooccurring L. monocytogenes strains in the samples. [ABSTRACT FROM AUTHOR]

Abstract Background Viral relapse is a major concern in hepatitis C virus (HCV) antiviral therapy. Currently, there are no satisfactory methods to predict viral relapse, especially in the era of direct acting antivirals in which the virus often quickly becomes undetectable using PCR-based approaches that focus on a small viral region. Next-generation sequencing (NGS) provides an alternative option for viral detection in a genome-wide manner. However, owing to the overwhelming dominance of human genetic content in clinical specimens, direct detection of HCV by NGS has a low sensitivity and hence viral enrichment is required. Methods Based on template-dependent multiple displacement amplification (tdMDA), an improved method for whole genome amplification (Wang et al., 2017. Biotechniques 63, 21–27), we evaluated two strategies to enhance the sensitivity of NGS-based HCV detection: duplex-specific nuclease (DSN)-mediated depletion of human sequences and HCV probe-based capture sequencing. Results In DSN-mediated depletion, human sequences were significantly reduced in the two HCV serum samples tested, 65.3% → 55.6% → 33.7% (#4727) and 68.6% → 56% → 21% (#4970), respectively for no normalization, self- and driver-applied normalization. However, this approach was associated with a loss of HCV sequences perhaps due to its micro-homology with the human genome. In capture sequencing, HCV-mapped sequencing reads occupied 96.8% (#4727) and 22.14% (#4970) in NGS data, equivalent to 1936x and 7380x enrichment, respectively. Capture sequencing was then applied to ten serum samples collected at the end of HCV antiviral therapy. Interestingly, the number of HCV-mapped reads was significantly higher in relapsed patients (n = 5) than those from patients with sustained virological response (SVR) (n = 5), 102.4 ± 72.3 vs. 2.6 ± 0.55, p = 0.014. Conclusions Our data provides concept evidence for a highly sensitive HCV detection by capture sequencing. The abundance difference of HCV sequencing reads at the end of HCV antiviral therapy could be applied to predict treatment outcomes.

Multiple displacement amplification (MDA) is a popular single-cell whole-genome amplification (WGA) technique that can greatly improve the amplification efficiency of single-cell genomes. However, there is an inherent problem that cannot be completely solved, that is, the amplification bias. We here propose an improved MDA method based on low melting agarose gel, named gelMDA. Firstly, the agarose gel and solution were characterized with SEM and fluorescent reagent. Then, we used gelMDA for cDNA amplification in library preparation of RNA-seq, and conventional MDA was used as a comparison. The sensitivity, efficiency of gelMDA, and amplification bias were evaluated with fluorescence curve, product yield, and the sequencing results. Finally, gelMDA was used for single-cell transcriptome sequencing. The results showed that the sensitivity and product yield of gelMDA were significantly higher than those of conventional MDA. A lower coefficient of variation (CV) and a higher reproducibility were obtained from gelMDA sequencing results. A region of 30 μm in diameter was amplified from the tissue sections and successfully sequenced. In conclusion, gelMDA obtained higher amplification efficiency and lower amplification bias in the present study. It suggested the great potential in single-cell RNA amplification and sequencing. [ABSTRACT FROM AUTHOR]

As the vast majority of microorganisms have yet to be cultivated in a laboratory setting, access to their genetic makeup has largely been limited to cultivation-independent methods. These methods, namely metagenomics and more recently single-cell genomics, have become cornerstones for microbial ecology and environmental microbiology. One ultimate goal is the recovery of genome sequences from each cell within an environment to move toward a better understanding of community metabolic potential and to provide substrate for experimental work. As single-cell sequencing has the ability to decipher all sequence information contained in an individual cell, this method holds great promise in tackling such challenge. Methodological limitations and inherent biases however do exist, which will be discussed here based on environmental and benchmark data, to assess how far we are from reaching this goal.

Transboundary emissions of smoke-haze from land and forest fires have recurred annually during the dry period (June to October, over the past few decades) in South East Asia. Hazardous air quality has been recorded in Malaysia during these episodes. Agricultural practices such as slash-and-burn of biomass and peat fires particularly in Sumatera and Kalimantan, Indonesia, have been implicated as the major causes of the haze. Past findings have shown that a diversity of microbes can thrive in air including in smoke-haze polluted air. In this study, metagenomic data were generated to reveal the diversity of microorganisms in air during days with and without haze. Air samples were collected during non-haze (2013A01) and two haze (2013A04 and 2013A05) periods in the month of June 2013. DNA was extracted from the samples, subjected to Multiple Displacement Amplification and whole genome sequencing (Next Generation Sequencing) using the HiSeq 2000 Platform. Extensive bio-informatic analyses of the raw sequence data then followed. Raw reads from these six air samples were deposited in the NCBI SRA databases under Bioproject PRJNA662021 with accession numbers SRX9087478, SRX9087479 and SRX9087480.

Objective To evaluate the efficiency of REPLI-g?? Single Cell Kit for sample DNA amplification, and explore its application value in forensic trace DNA amplification. Methods Three DNA extraction kits were selected to extract DNA from peripheral blood of 10 unrelated individuals. The DNA yield and purity of the three DNA extraction kits were compared. According to the results of comparison, one DNA sample was selected to concentrate and dilute, then used as the initial sample of whole genome amplification （WGA）. REPLI-g?? Single Cell Kit was used to amplify the initial sample at the whole genome level. The amplification yield and amplification times were calculated, and the distribution of DNA fragments was detected by agarose gel electrophoresis. GoldeneyeTM DNA ID System 20A Kit was used to perform the STR typing of the initial sample and DNA samples amplified at the whole genome level to evaluate the performance of REPLI-g?? Single Cell Kit in trace DNA amplication in terms of purity and yield as well as the success rate of STR typing. Results After comparison, one DNA sample was selected from QIAsymphony?? DNA Investigator?? Kit extracts to concentrate and dilute as the initial sample of WGA. After amplifying the whole genome of a series of initial samples by REPLI-g?? Single Cell Kit, the lowest average of amplification yield reached 8.77×103 ng, while the average of the corresponding amplification times reached 1.40×106. DNA fragments were large and concentrated. The STR typing success rate of WGA samples became lower with the decrease of initial samples used, but when the amount of samples was lower than 0.5 ng, the STR typing success rate of samples after DNA WGA was higher than that of samples without DNA WGA. Conclusion REPLI-g?? Single Cell Kit can increase the yield of template DNA. Especially for trace DNA, the STR typing success rate can be improved to a certain extent.

Recent advances in single-cell genomics provide an alternative to largely gene-centric metagenomics studies, enabling whole-genome sequencing of uncultivated bacteria. However, single-cell assembly projects are challenging due to (i) the highly nonuniform read coverage and (ii) a greatly elevated number of chimeric reads and read pairs. While recently developed single-cell assemblers have addressed the former challenge, methods for assembling highly chimeric reads remain poorly explored. We present algorithms for identifying chimeric edges and resolving complex bulges in de Bruijn graphs, which significantly improve single-cell assemblies. We further describe applications of the single-cell assembler SPAdes to a new approach for capturing and sequencing "microbial dark matter" that forms small pools of randomly selected single cells (called a mini-metagenome) and further sequences all genomes from the mini-metagenome at once. On single-cell bacterial datasets, SPAdes improves on the recently developed E+V-SC and IDBA-UD assemblers specifically designed for single-cell sequencing. For standard (cultivated monostrain) datasets, SPAdes also improves on A5, ABySS, CLC, EULER-SR, Ray, SOAPdenovo, and Velvet. Thus, recently developed single-cell assemblers not only enable single-cell sequencing, but also improve on conventional assemblers on their own turf. SPAdes is available for free online download under a GPLv2 license.

Abstract Background The field of diagnostics continues to advance rapidly with a variety of novel approaches, mainly dependent upon high technology platforms. Nonetheless much diagnosis, particularly in developing countries, still relies upon traditional methods such as microscopy. Biological material, particularly nucleic acids, on archived glass slides is a potential source of useful information both for diagnostic and epidemiological purposes. There are significant challenges faced when examining archived samples in order that an adequate amount of amplifiable DNA can be obtained. Herein, we describe a model system to detect low numbers of bacterial cells isolated from glass slides using (laser capture microscopy) LCM coupled with PCR amplification of a suitable target. Results Mycobacterium smegmatis was used as a model organism to provide a proof of principle for a method to recover bacteria from a stained sample on a glass slide using a laser capture system. Ziehl-Neelsen (ZN) stained cells were excised and catapulted into tubes. Recovered cells were subjected to DNA extraction and pre-amplified with multiple displacement amplification (MDA). This system allowed a minimum of 30 catapulted cells to be detected following a nested real-time PCR assay, using rpoB specific primers. The combination of MDA and nested real-time PCR resulted in a 30-fold increase in sensitivity for the detection of low numbers of cells isolated using LCM. Conclusions This study highlights the potential of LCM coupled with MDA as a tool to improve the recovery of amplifiable nucleic acids from archived glass slides. The inclusion of the MDA step was essential to enable downstream amplification. This platform should be broadly applicable to a variety of diagnostic applications and we have used it as a proof of principle with a Mycobacterium sp. model system.

Multiple displacement amplification (MDA) is a popular whole genome amplification method for its simplicity and high coverage on whole genome DNA. Yet it suffers from uneven magnification and long reaction time. Here we present the cyclical concentration adjusting concept to speed up MDA and realize the droplet dilution steps with a novel droplet re-generation strategy. The increased initial concentration shortened inefficient time and the repeated high-speed period maintained the reaction efficient for long. This rapid droplet MDA method could save more than 1/2 of the total reaction time and exhibited higher accuracy than the conventional method. The cyclical concentration adjusting method alters nothing but the concentration of reactants in different periods of reaction. Therefore, it may be widely utilized to accelerating MDA-like time-consuming reactions without concerns of compatibility. Image 1 • Halving the waiting time of multiple displacement amplification (MDA) with the cyclical concentration adjusting strategy. • Accelerating principle explained and saving time can be estimated accordingly. • High throughput droplet dilution with the droplet regeneration method. • Superior fidelity and coverage of amplified samples. [ABSTRACT FROM AUTHOR]

Symbioses between microbes are likely widespread and functionally relevant in diverse biological systems; however, they are difficult to discover. Most microbes remain uncultivated, symbioses can be relatively rare or dynamic, and intercellular connections can be delicate. Thus, traditional methods such as microscopy are inadequate for efficient discovery and precise characterization of novel interactions, their metabolic basis, and the species involved. High-throughput metagenomic sequencing of entire microbial communities has revolutionized the field of microbial ecology; however, genomic signals from symbionts can get buried in sequences from abundant organisms and evidence for direct links between microbial species cannot be gained from bulk samples. Thus, a specialized approach to the characterization of symbioses between naturally occurring microbes is required. This chapter presents methods for combining fluorescence-activated cell sorting to isolate and separate uncultivated symbionts with molecular biology techniques for DNA amplification in order to characterize uncultivated symbionts through genomic and metagenomic techniques.

Symbioses between microbes are likely widespread and functionally relevant in diverse biological systems; however, they are difficult to discover. Most microbes remain uncultivated, symbioses can be relatively rare or dynamic, and intercellular connections can be delicate. Thus, traditional methods such as microscopy are inadequate for efficient discovery and precise characterization of novel interactions, their metabolic basis, and the species involved. High-throughput metagenomic sequencing of entire microbial communities has revolutionized the field of microbial ecology; however, genomic signals from symbionts can get buried in sequences from abundant organisms and evidence for direct links between microbial species cannot be gained from bulk samples. Thus, a specialized approach to the characterization of symbioses between naturally occurring microbes is required. This chapter presents methods for combining fluorescence-activated cell sorting to isolate and separate uncultivated symbionts with molecular biology techniques for DNA amplification in order to characterize uncultivated symbionts through genomic and metagenomic techniques.

Combining multiple displacement amplification (MDA) with metagenomics enables the analysis of samples with extremely low DNA concentrations, making them suitable for high-throughput sequencing. Although amplification bias and nonspecific amplification have been reported from MDA-amplified samples, the impact of MDA on metagenomic datasets is not well understood. We compared three MDA methods (i.e. bulk MDA, emulsion MDA, and primase MDA) for metagenomic analysis of two DNA template concentrations (approx. 1 and 100 pg) derived from a microbial community standard "mock community" and two low biomass environmental samples (i.e. borehole fluid and groundwater). We assessed the impact of MDA on metagenome-based community composition, assembly quality, functional profiles, and binning. We found amplification bias against high GC content genomes but relatively low nonspecific amplification such as chimeras, artifacts, or contamination for all MDA methods. We observed MDA-associated representational bias for microbial community profiles, especially for low-input DNA and with the primase MDA method. Nevertheless, similar taxa were represented in MDA-amplified libraries to those of unamplified samples. The MDA libraries were highly fragmented, but similar functional profiles to the unamplified libraries were obtained for bulk MDA and emulsion MDA at higher DNA input and across these MDA libraries for the groundwater sample. Medium to low-quality bins were possible for the high input bulk MDA metagenomes for the most simple microbial communities, borehole fluid, and mock community. Although MDA-based amplification should be avoided, it can still reveal meaningful taxonomic and functional information from samples with extremely low DNA concentration where direct metagenomics is otherwise impossible., Competing Interests: The authors declare that they have no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Background: The extremely small amount of DNA in a cell makes it difficult to study the whole genome of single cells, so whole-genome amplification (WGA) is necessary to increase the DNA amount and enable downstream analyses. Multiple displacement amplification (MDA) is the most widely used WGA technique. Results: Compared with amplification methods based on PCR and other methods, MDA renders high-quality DNA products and better genome coverage by using phi29 DNA polymerase. Moreover, recently developed advanced MDA technologies such as microreactor MDA, emulsion MDA, and micro-channel MDA have improved amplification uniformity. Additionally, the development of other novel methods such as TruePrime WGA allows for amplification without primers. Conclusion: Here, we reviewed a selection of recently developed MDA methods, their advantages over other WGA methods, and improved MDA-based technologies, followed by a discussion of future perspectives. With the continuous development of MDA and the successive update of detection technologies, MDA will be applied in increasingly more fields and provide a solid foundation for scientific research. [ABSTRACT FROM AUTHOR]

Rare members of environmental microbial communities are often overlooked and unexplored, primarily due to the lack of techniques capable of acquiring their genomes. Chloroflexi belong to one of the most understudied phyla, even though many of its members are ubiquitous in the environment and some play important roles in biochemical cycles or biotechnological applications. We here used a targeted cell-sorting approach, which enables the selection of specific taxa by fluorescent labeling and is compatible with subsequent single-cell genomics, to enrich for rare Chloroflexi species from a wastewater-treatment plant and obtain their genomes. The combined workflow was able to retrieve a substantially higher number of novel Chloroflexi draft genomes with much greater phylogenetical diversity when compared to a metagenomics approach from the same sample. The method offers an opportunity to access genetic information from rare biosphere members which would have otherwise stayed hidden as microbial dark matter and can therefore serve as an essential complement to cultivation-based, metagenomics, and microbial community-focused research approaches.

Development of potential HIV-1 curative interventions requires accurate characterization of the proviral reservoir, defined as host-integrated viral DNA genomes that drive rebound of viremia upon halting ART (antiretroviral therapy). Evaluation of such interventions necessitates methods capable of pinpointing the rare, genetically intact, replication-competent proviruses within a background of defective proviruses. This evaluation can be achieved by identifying the distinct integration sites of intact proviruses within host genomes and monitoring the dynamics of these proviruses and host cell lineages over longitudinal sampling. Until recently, molecular genetic approaches at the single proviral level have been generally limited to one of a few metrics, such as proviral genome sequence/intactness, host-proviral integration site, or replication competency. New approaches, taking advantage of MDA (multiple displacement amplification) for WGA (whole genome amplification), have enabled multiparametric proviral characterization at the single-genome level, including proviral genome sequence, host-proviral integration site, and phenotypic characterization of the host cell lineage, such as CD4 memory subset and antigen specificity. In this review, we will examine the workflow of MDA-augmented molecular genetic approaches to study the HIV-1 reservoir, highlighting technical advantages and flexibility. We focus on a collection of recent studies in which investigators have used these approaches to comprehensively characterize intact and defective proviruses from donors on ART, investigate mechanisms of elite control, and define cell lineage identity and antigen specificity of infected CD4+ T cell clones. The highlighted studies exemplify how these approaches and their future iterations will be key in defining the targets and evaluating the impacts of HIV curative interventions.

Rare members of environmental microbial communities are often overlooked and unexplored, primarily due to the lack of techniques capable of acquiring their genomes. Chloroflexi belong to one of the most understudied phyla, even though many of its members are ubiquitous in the environment and some play important roles in biochemical cycles or biotechnological applications. We here used a targeted cell-sorting approach, which enables the selection of specific taxa by fluorescent labeling and is compatible with subsequent single-cell genomics, to enrich for rare Chloroflexi species from a wastewater-treatment plant and obtain their genomes. The combined workflow was able to retrieve a substantially higher number of novel Chloroflexi draft genomes with much greater phylogenetical diversity when compared to a metagenomics approach from the same sample. The method offers an opportunity to access genetic information from rare biosphere members which would have otherwise stayed hidden as microbial dark matter and can therefore serve as an essential complement to cultivation-based, metagenomics, and microbial community-focused research approaches. [ABSTRACT FROM AUTHOR]

Multiple displacement amplification (MDA) is widely used in whole-genome/transcriptome amplification. However, template-independent amplification (TIA) in MDA is a commonly observed phenomenon, particularly when using high concentrations of random hexamer primers and extended incubation times. Here, we demonstrate that the use of random pentamer primers with 5´ ends blocked by a C18 spacer results in MDA solely in a template-dependent manner, a technique we have named tdMDA. Together with an optimized procedure for the removal of residual genomic DNA during RNA extraction, tdMDA was used to profile circulating RNA from 0.2 mL of patient sera. In comparison to regular MDA, tdMDA demonstrated a lack of quantifiable DNA amplification in the negative control, a remarkable reduction of unmapped reads from Illumina sequencing (7 ± 10.9% versus 58.6 ± 39%, P = 0.006), and increased mapping rates of the serum transcriptome (26.9 ± 7.9% versus 5.8 ± 8.2%, P = 3.8 × 10-4). Transcriptome profiles could be used to separate patients with chronic hepatitis C virus (HCV) infection from those with HCV-associated hepatocellular carcinoma (HCC). We conclude that tdMDA should facilitate RNA-based liquid biopsy, as well as other genome studies with biological specimens having ultralow amounts of genetic material.

Abstract Background Sequencing analysis of circulating tumor cells (CTCs) enables “liquid biopsy” to guide precision oncology strategies. However, this requires low-template whole genome amplification (WGA) that is prone to errors and biases from uneven amplifications. Currently, quality control (QC) methods for WGA products, as well as the number of CTCs needed for reliable downstream sequencing, remain poorly defined. We sought to define strategies for selecting and generating optimal WGA products from low-template input as it relates to their potential applications in precision oncology strategies. Methods Single pancreatic cancer cells (HPAF-II) were isolated using laser microdissection. WGA was performed using multiple displacement amplification (MDA), multiple annealing and looping based amplification (MALBAC) and PicoPLEX. Quality of amplified DNA products were assessed using a multiplex/RT-qPCR based method that evaluates for 8-cancer related genes and QC-scores were assigned. We utilized this scoring system to assess the impact of de novo modifications to the WGA protocol. WGA products were subjected to Sanger sequencing, array comparative genomic hybridization (aCGH) and next generation sequencing (NGS) to evaluate their performances in respective downstream analyses providing validation of the QC-score. Results Single-cell WGA products exhibited a significant sample-to-sample variability in amplified DNA quality as assessed by our 8-gene QC assay. Single-cell WGA products that passed the pre-analysis QC had lower amplification bias and improved aCGH/NGS performance metrics when compared to single-cell WGA products that failed the QC. Increasing the number of cellular input resulted in improved QC-scores overall, but a resultant WGA product that consistently passed the QC step required a starting cellular input of at least 20-cells. Our modified-WGA protocol effectively reduced this number, achieving reproducible high-quality WGA products from ≥5-cells as a starting template. A starting cellular input of 5 to 10-cells amplified using the modified-WGA achieved aCGH and NGS results that closely matched that of unamplified, batch genomic DNA. Conclusion The modified-WGA protocol coupled with the 8-gene QC serve as an effective strategy to enhance the quality of low-template WGA reactions. Furthermore, a threshold number of 5–10 cells are likely needed for a reliable WGA reaction and product with high fidelity to the original starting template.

Aim: To evaluate whether using multiple displacement amplification (MDA) as the first step can increase the diagnostic efficiency of preimplantation genetic testing for monogenic disease (PGT‐M) for β‐thalassemia. Methods: This is a retrospective cohort study. All included patients underwent PGT‐M cycles (n = 307) for β‐thalassemia in our center from January 2014 to February 2018. We divided the patients into two groups based on two different detection methods. For the polymerase chain reaction (PCR) group (n = 115), multiplex nested PCR+ reverse dot blot analysis was performed directly after cell lysis. For the MDA group (n = 192), the whole genomes of single cells were directly amplified using MDA and then examined by singleplex PCR + reverse dot blot for β‐thalassemia. Results: A total of 2315 embryos were tested. The overall diagnostic efficiency of the MDA group was significantly higher than that of the PCR group (96.99% vs 88.15%, P < 0.001). The percentage of embryos available for transfer was significantly higher in the MDA group than in the PCR group (74.28% vs 64.98%, P < 0.001). Furthermore, the carrier embryo rate of the MDA group was significantly higher than that of the PCR group (50.11% vs 35.95%, P < 0.001). Conclusion: This study indicates that MDA, as the first step in PGT‐M for β‐thalassemia, can increase diagnostic efficiency. [ABSTRACT FROM AUTHOR]

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Whole genome amplification (WGA) is crucial for whole genome sequencing to investigate complex genomic alteration at the single-cell or even single-molecule level. Multiple displacement amplification (MDA) and multiple annealing and looping based amplification cycles (MALBAC) are two most widely applied WGA methods, which have different advantages and disadvantages, dependent on research objectives. Herein, we compared the MDA and MALBAC to provide more information on their performance in droplets and tubes. We observed that the droplet method could dramatically reduce the amplification bias and retain the high accuracy of replication than the conventional tube method. Furthermore, the droplet method exhibited higher efficiency and sensitivity for both homozygous and heterozygous single nucleotide variants (SNVs) at the low sequencing depth. In addition, we also found that MALBAC offered a greater uniformity and reproducibility and MDA showed a better efficiency of genomic coverage and SNV detection. Our results provided insights that will allow future decision making.

Fast point-of-use detection of, for example, early-stage zoonoses, e.g., Q-fever, bovine tuberculosis, or the Covid-19 coronavirus, is beneficial for both humans and animal husbandry as it can save lives and livestock. The latter prevents farmers from going bankrupt after a zoonoses outbreak. This paper describes the development of a fabrication process and the proof-of-principle of a disposable DNA amplification chip with an integrated heater. Based on the analysis of the milling process, metal adhesion studies, and COMSOL MultiPhysics heat transfer simulations, the first batch of chips has been fabricated and successful multiple displacement amplification reactions are performed inside these chips. This research is the first step towards the development of an early-stage zoonoses detection device. Tests with real zoonoses and DNA specific amplification reactions still need to be done.