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Rolling Circle Amplification (RCA) and Bridge Amplification are two distinct amplification techniques used in molecular biology for DNA amplification. Each method has its own principles, applications, and advantages. Here’s a detailed comparison of the two:

Rolling Circle Amplification (RCA)

Definition: Rolling Circle Amplification is a method that amplifies circular DNA templates to produce long, repetitive strands of DNA.

Key Features:

– Mechanism: RCA involves the use of a circular DNA template. A DNA polymerase enzyme binds to the circular template and synthesizes a long strand of DNA by continuously adding nucleotides. This process “rolls” around the circular template, creating a long single-stranded DNA product that can be converted into double-stranded DNA.

– Template: The starting material is typically a circular DNA molecule, such as a plasmid or a specially designed circular oligonucleotide.

– Output: The output is a high yield of DNA, often in the form of concatamers (long chains of repeated sequences).

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Bridge Amplification

Definition: Bridge Amplification is a method used primarily in next-generation sequencing (NGS) and microarray applications to amplify DNA fragments on a solid surface.

Key Features:

– Mechanism: In bridge amplification, single-stranded DNA fragments are attached to a solid surface (such as a flow cell). The DNA is then amplified through a series of cycles where the strands are hybridized to complementary oligonucleotides on the surface. The DNA polymerase extends the strands, creating a “bridge” structure. This process results in clusters of identical DNA fragments.

– Template: The starting material is typically linear, single-stranded DNA fragments.

– Output: The output is clusters of amplified DNA, which can be sequenced or analyzed.

Applications:

– Next-Generation Sequencing (NGS): Widely used in sequencing platforms like Illumina.

– Microarray Technology: Used for analyzing gene expression and genotyping.

In the context of DNA sequencing, Read 1 and Read 2 refer to the two sequencing reads generated from a DNA fragment during the sequencing process. Here’s a breakdown of each:

Read 1:

– Definition: Read 1 is the first sequence obtained from a DNA fragment during the sequencing process.

– Process: During sequencing, the DNA is prepared and amplified, and then the first strand of the DNA is sequenced. This involves incorporating reversible terminator nucleotides, capturing the fluorescent signal, and determining the sequence of bases in that strand.

– Importance: Read 1 provides the initial sequence information, which is crucial for identifying the DNA fragment and for downstream analysis.

Read 2:

– Definition: Read 2 is the complementary sequence obtained after Read 1 is completed.

– Process: After Read 1 is finished, the sequencing process continues by using the complementary strand of the DNA fragment. The reversible terminator nucleotides are replaced with regular dNTPs, and the sequencing polymerase extends the complementary strand to generate Read 2.

– Importance: Read 2 enhances the accuracy of the sequencing results by providing information from both strands of the DNA fragment. This dual-read approach helps in resolving ambiguities and improving the overall quality of the sequencing data.

Summary:

The combination of Read 1 and Read 2 allows for paired-end sequencing, which is a powerful method that increases the accuracy and reliability of the sequencing results. This approach is particularly beneficial for applications such as genome assembly, variant detection, and structural variation analysis , .

MDA stands for Multiple Displacement Amplification. It is a technique used to amplify DNA, particularly useful in scenarios where the starting material is limited, such as in single-cell genomics or when working with degraded samples.

Significance of MDA:

1. High Sensitivity:

MDA can amplify minute amounts of DNA, making it ideal for applications where only small quantities of DNA are available.

2. Uniform Amplification:

The method provides a more uniform amplification of the entire genome compared to other amplification methods, which can lead to better representation of the original DNA.

3. Application in Sequencing:

In the context of sequencing, MDA is used to generate sufficient quantities of DNA for sequencing libraries. After the initial sequencing read (Read1), MDA polymerase is employed to extend the DNA strands, allowing for further sequencing (Read2) and enhancing the overall signal during the sequencing process .

Overall, MDA is a crucial technique in genomic research, enabling the analysis of DNA from limited or challenging samples.

The sequencing principle involves several key components, each playing a crucial role in the overall process. Here are the significant components and their functions:

1. DNA Nanoballs (DNBs):

These are formed during the library preparation and amplification process. DNBs consist of multiple copies of the same DNA fragment, which enhances the signal during sequencing by providing a higher concentration of the target DNA .

2. Reversible Terminator Nucleotides:

Initially used in the sequencing process, these nucleotides allow for the incorporation of a single base at a time. After each incorporation, the fluorescent signal is captured, and the terminator is removed to allow the next base to be added. This step is crucial for accurate base calling during sequencing .

3. MDA Polymerase:

This enzyme is used during the amplification phase after the first read (Read1) is completed. It replaces the reversible terminator nucleotides with regular dNTPs, allowing for the continuous extension of the DNA strand. This step is essential for generating sufficient DNA for the second read (Read2) and for the overall amplification of the library , .

4. Read1 and Read2:

These refer to the two sequencing reads that are generated. Read1 is the first sequence obtained, while Read2 is the complementary sequence obtained after the first read. The ability to read both strands of the DNA fragment increases the accuracy and reliability of the sequencing results .

5. Index Read:

After completing Read1 and Read2, an index read is performed. This step allows for the identification of different samples in multiplexed sequencing runs, enabling multiple samples to be sequenced simultaneously and efficiently .

Each of these components is integral to the sequencing workflow, ensuring that the process is efficient, accurate, and capable of generating high-quality genomic data.

1. Fragmentation of DNA:

The genomic DNA (gDNA) is first fragmented to obtain the desired size for sequencing.

2. End Repair and A-tailing:

The fragmented DNA undergoes end repair to create blunt ends, followed by the addition of an ‘A’ base to the 3′ ends to facilitate adapter ligation.

3. Adapter Ligation:

Specific adapters are ligated to the ends of the DNA fragments. This step is crucial for the subsequent amplification and sequencing processes.

4. PCR Amplification:

The library is then amplified using PCR to enrich the fragments that have successfully ligated adapters. This step typically focuses on amplifying the positive strand of the library , .

5. Purification:

The amplified library is purified to remove any unligated adapters and other contaminants.

6. Quality Control:

The quality and quantity of the library are assessed to ensure it meets the requirements for sequencing.

These steps collectively prepare the gDNA library for the sequencing process, ensuring that the DNA fragments are suitable for high-throughput sequencing technologies .

Comparison with Reference Data

The authors conditionally considered the Illumina HiSeq 2500 data as a standard reference to evaluate the accuracy of the MGISEQ-2000 data. This involved calculating “error rates” such as “False Positive” and “False Negative” rates in the MGISEQ-2000 dataset (E704-M) using the Illumina dataset (E704-I) as a benchmark .

Variant Calling Analysis

The authors utilized multiple software packages for variant calling, including Strelka2, to analyze the datasets generated by both platforms. They reported the total number of SNPs detected, the sensitivity of SNP determination, and the false positive rate (FPR) for the MGISEQ-2000 relative to the Illumina data. The sensitivity for SNP detection in the MGISEQ-2000 sample was found to be 99.51%, with an FPR of 0.000254% .

F1 Metrics Calculation

They calculated the F1 metric, which is a measure of a test’s accuracy that considers both precision and recall. For SNPs, the F1 metric was reported as 99.65%, indicating high accuracy in SNP detection for the MGISEQ-2000 platform .

Indel Detection Accuracy

Similar assessments were made for indel detection, where the sensitivity was reported as 98.84% with an F1 metric of 98.81%. This further demonstrated the reliability of the MGISEQ-2000 in detecting genomic variants .

Through these methods, the authors were able to conclude that the MGISEQ-2000 platform provided a high level of accuracy in SNP detection, comparable to that of the Illumina HiSeq 2500.

Source:

https://doi.org/10.1371/journal.pone.0230301

Duplication Rates

The MGISEQ-2000 exhibited a lower duplication rate compared to the Illumina HiSeq 2500 when analyzing individual fastq files. The overall duplication rate for the Illumina platform was higher (12.26%) due to the merging of fastq files from two different flow cells, while the duplication rate for both platforms did not exceed 5-6% in individual files.

Data Generation and Coverage

Both platforms generated comparable amounts of data, with MGISEQ-2000 producing 101.37 Gb and HiSeq 2500 producing 94.37 Gb. The average coverage was also similar, with MGISEQ-2000 at 32.75X and HiSeq 2500 at 30.48X, indicating that both platforms provide similar sequencing quality.

Error Rates and Variant Detection

The study found that while the MGISEQ-2000 had slightly inferior performance in terms of random sequencing errors and indel detection accuracy, the differences were small and generally insignificant for most research tasks. The detection rates of genomic variants were similar between the two platforms, suggesting that they can be used interchangeably for many applications,.

Library Preparation Methods

Different methods of DNA fragmentation were used for library preparation, with MGISEQ-2000 utilizing ultrasound fragmentation and Illumina HiSeq 2500 using enzymatic fragmentation. This difference in preparation methods is important for interpreting the results.

Overall, while both platforms demonstrated comparable performance in many aspects, the MGISEQ-2000 can be considered a viable alternative to the Illumina HiSeq 2500 for whole-genome sequencing tasks.


Source:

https://doi.org/10.1371/journal.pone.0230301

To identify the sequence in DNA, each base in a DNA fragment or DNA nanoball needs to be identified step-by-step. First, a primer binds to the adaptor in the DNA strand, and the primer will be extended using an artificial dNTP rather than a natural dNTP. The artificial dNTP usually prevents further extension, so one base is added at a time. That base could be identified as A, T, C, or G by detecting the unique fluorescent dye (unique for each base) on it, with a camera. Another technology, which is called coolMPS, detects the dye on the monoclonal antibodies. Each type of the monoclonal antibodies binds to a specific base. Anyway, a reversible terminator is often needed in these technologies (second-generation sequencing) to ensure that only one base is added at a time.

How is it done? DNA extension by DNA polymerase requires a 3’-OH group in the last base (3’-end). The group can be replaced with a different chemical group, preventing the extension. But the artificial 3’-O-blocking group can be reacted to specific reagent to be reverted into an OH group to allow extension again. The dye will also be cleaved away to remove the signal responsible for this base. The cycle repeats to identity the next bases.

MGI-seq or DNB-seq is a next-generation sequencing (NGS) platform developed by MGI Tech Co., Ltd. It could perform whole-genome sequencing (WGS), sequencing of cell-free DNA fragments (cfDNA), ATAC-Seq, etc. It is also a form of sequencing by synthesis (SBS) technology. But different from Illumina sequencing which utilizes bridge amplification, DNB-Seq requires the rolling circle amplification of circular DNA to form DNA nanoball (DNB) for library preparation.

Let’s introduce the library preparation steps, that could be done manually or with automation platforms, for example, MGISP-100, MGISP-960. For WGS, the genomic DNA is fragmented. The fragments are end-repaired into 5’phosphorylated blunt-ended DNA, size-selected with magnetic beads, then ligated to adaptor pairs. After cleanup (repurification), the two ends of the DNA fragmented are ligated to form circular single-stranded DNA (ssDNA). The products (either the product DNA fragments or the DNB library below) from multiple samples could be pooled together for sequencing, while each sample having different barcodes (index).

DNA nanoballs (DNB) are produced by rolling circle amplification of the circular ssDNA, forming a clump of long ssDNA. The DNBs are then loaded and hybridized into the positively charged spots on a patterned array on the flowcell (chip). Sequencing process could proceed with the sequencing machine, such as DNBSEQ-T7, DNBSEQ-G400, DNBSEQ-G99, etc. The sequencing mode is also divided into single-end or paired-end sequencing (e.g., SE50, PE150). And in sequencing-by-synthesis, labeled antibodies that bind specific nucleotide with reversible terminator can be used to identify the sequence rather than using fluorescent-labeled dNTPs. This is called CoolMPS technology. See more details in later posts.To access the accuracy of the sequencing data, the quality score Q30 (%) is often used, with 100% being the best score. Q40 is the upcoming quality score.

The team has been trying to develop the best DNA polymerase mutants, MDA (multiple displacement amplification) enzymes, better dNTP designs, etc., to improve this fantastic technology.