Chimera: The spoiler in multiple displacement amplification

Lü, Na; Qiao, Yi; Zhang, Lu; Tu, Jing

doi:10.1016/j.csbj.2023.02.034

Cited by 6 publications

(14 citation statements)

References 85 publications

(165 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…If there isn't sufficient coverage obtained post-CADECT run, an alternative is to merge the reads identified as short by the program with the nonconcatemeric reads. As observed previously, the chimeric rate produced by MDA is positively associated with the mean read length (Lu et al 2023), indicating a decreased likelihood of chimeric reads in this short dataset. Consequently, this dataset is less likely to negatively impact the assembly process.…”

Section: Discussionsupporting

confidence: 81%

“…3A). While the occurrence of concatemers in MDA assays has been reported previously (Paul and Apgar 2005;Lu et al 2023), they are typically present in low amounts after sequencing.…”

Section: Mda Results In An Unexpected Size Increase From Fragmented D...mentioning

confidence: 71%

“…These include: (i) Nonspecific amplification resulted from primer dimer formation causing template switching or contamination by DNA templates; (ii) Formation of chimeric DNA rearrangements; and (iii) Representation bias, which can affect the accuracy and completeness of the amplified genomic material (Binga et al 2008). Some studies shows that chimeric reads are usually invert chimeras or direct chimeras, but it was previously observed that most of detected MDA chimeric sequences (85%) are inverted chimeras, such as inverted sequences with intervening deletions which can be caused by template switching (Lasken and Stockwell 2007; Lu et al 2023). These chimeric sequences are known to affect genome sequencing since they can be considered as amplification artifacts, which cannot be used for genome assembling (Lu et al 2023).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

“Evaluating the Benefits and Limits of Multiple Displacement Amplification with Whole-Genome Oxford Nanopore Sequencing”

Dadzie,

Beaudry,

Deyanov

et al. 2024

Preprint

View full text Add to dashboard Cite

Multiple Displacement Amplification (MDA) outperforms conventional PCR in long fragment and whole genome amplification which makes it attractive to couple with long-read sequencing of samples with limited quantities of DNA to obtain improved genome assemblies. Here, we explore the efficacy and limits of MDA for genome sequence assembly using Oxford Nanopore Technologies (ONT) rapid library preparations and minION sequencing. We successfully generated almost complete genome sequences for all organisms examined, including Cryptosporidium meleagridis, Staphylococcus aureus, Enterococcus faecium, and Escherichia coli, with the ability to generate high-quality data from samples starting with only 0.025 ng of total DNA. Controlled sheared DNA samples exhibited a distinct pattern of size-increase after MDA, which may be associated with the amplification of long, low-abundance fragments present in the assay, as well as generating concatemeric sequences during amplification. To address concatemers, we developed a computational pipeline (CADECT: Concatemer Detection Tool) to identify and remove putative concatemeric sequences. This study highlights the efficacy of MDA in generating high-quality genome assemblies from limited amounts of input DNA. Also, the CADECT pipeline effectively mitigated the impact of concatemeric sequences, enabling the assembly of contiguous sequences even in cases where the input genomic DNA was degraded. These results have significant implications for the study of organisms that are challenging to culture in vitro, such as Cryptosporidium, and for expediting critical results in clinical settings with limited quantities of available genomic DNA.

show abstract

Section: Discussionsupporting

confidence: 81%

“…3A). While the occurrence of concatemers in MDA assays has been reported previously (Paul and Apgar 2005;Lu et al 2023), they are typically present in low amounts after sequencing.…”

Section: Mda Results In An Unexpected Size Increase From Fragmented D...mentioning

confidence: 71%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

“Evaluating the Benefits and Limits of Multiple Displacement Amplification with Whole-Genome Oxford Nanopore Sequencing”

Dadzie,

Beaudry,

Deyanov

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

“…Phi29 polymerase is known to produce complex hyper-branched structures and chimeras in amplified DNA (20). These artifacts can introduce DNA assembly errors in sequencing workflows (20). Thus, the 96 T. kodakarensis amplified fosmids were treated with T7 endonuclease I to remove branched DNA structures formed by the polymerase and prepared for multiplexed ONT sequencing using 96 barcodes as described in the Materials and Methods.…”

Section: Resultsmentioning

confidence: 99%

“…Amplified fosmids were first debranched using T7 endonuclease I to remove the branching DNA structures that are often created by phi29 (20). In a new 96-well PCR plate, 12 μL of each phi29-XT-amplified product were mixed with 1 μL of T7 endonuclease I, 2 μL of NEBuffer™ 2 and 5 μL of water.…”

Section: Methodsmentioning

confidence: 99%

High-throughput nanopore DNA sequencing of large insert fosmid clones directly from bacterial colonies

Chuzel,

Sinha,

Cunningham

et al. 2024

Preprint

View full text Add to dashboard Cite

Fosmids and cosmids are vectors frequently used in functional metagenomic studies. With a large insert capacity (around 30 kb) they can encode dozens of cloned genes or in some cases, entire biochemical pathways. Fosmids with cloned inserts can be transferred to heterologous hosts and propagated to enable screening for new enzymes and metabolites. After screening, fosmids from clones with an activity of interest must be de novo sequenced, a critical step towards identification of the gene(s) of interest. In this work, we present a new approach for rapid and high-throughput fosmid sequencing directly from Escherichia coli colonies without liquid culturing or fosmid purification. Our sample preparation involves fosmid amplification with phi29 polymerase and then direct nanopore sequencing using the Oxford Nanopore Technologies system. We also present a bioinformatics pipeline termed "phiXXer" that facilitates both de novo read assembly and vector trimming to generate a linear sequence of the fosmid insert. Finally, we demonstrate accurate sequencing of 96 fosmids in a single run and validate the method using two fosmid libraries that contain cloned large insert (~30-40 kb) genomic or metagenomic DNA.

show abstract

Recent Innovations and Technical Advances in High‐Throughput Parallel Single‐Cell Whole‐Genome Sequencing Methods

Qiao,

Cheng,

Miao

et al. 2024

Small Methods

View full text Add to dashboard Cite

Single‐cell whole‐genome sequencing (scWGS) detects cell heterogeneity at the aspect of genomic variations, which are inheritable and play an important role in life processes such as aging and cancer progression. The recent explosive development of high‐throughput single‐cell sequencing methods has enabled high‐performance heterogeneity detection through a vast number of novel strategies. Despite the limitation on total cost, technical advances in high‐throughput single‐cell whole‐genome sequencing methods are made for higher genome coverage, parallel throughput, and level of integration. This review highlights the technical advancements in high‐throughput scWGS in the aspects of strategies design, data efficiency, parallel handling platforms, and their applications on human genome. The experimental innovations, remaining challenges, and perspectives are summarized and discussed.

show abstract

Chimera: The spoiler in multiple displacement amplification

Cited by 6 publications

References 85 publications

“Evaluating the Benefits and Limits of Multiple Displacement Amplification with Whole-Genome Oxford Nanopore Sequencing”

“Evaluating the Benefits and Limits of Multiple Displacement Amplification with Whole-Genome Oxford Nanopore Sequencing”

High-throughput nanopore DNA sequencing of large insert fosmid clones directly from bacterial colonies

Recent Innovations and Technical Advances in High‐Throughput Parallel Single‐Cell Whole‐Genome Sequencing Methods

Contact Info

Product

Resources

About