2/28/2023 0 Comments Qctools github![]() The length distribution for the polymerase reads/subreads also provides a useful metric of run quality. Thus the number of productive ZMWs (ZMWs that received exactly one template) will directly indicate the productivity of the SMRTcell. All ZMWs within a SMRTcell are processed in parallel, sequencing thousands of templates at the same time. Each base incorporation will result in the release of a fluorophore, producing distinct light wavelengths per base. The template (SMRTBell) is introduced into the ZMW and nucleotide bases labeled with different fluorophores are sequentially added. These ZMWs contain a light-detection module and an immobilzed polymerase enzyme. Sequencing is performed within a SMRTcell which contains tens of thousands of zero-mode waveguides (ZMWs). Multiple copies of subreads generated from the single SMRTBell can then be collapsed to a single, high-quality sequence, called the “read of insert” or Circular Consensus Sequence (CCS). All other sequences sequenced from the CLR are called “scraps”. The CLRs are processed to remove adapter sequences and to retain only the insert sequence, called “subreads”. This CLR read may include sequence from adapters and multiple copies of inserts, because it traverses the circular template many times. The contiguous sequence generated by the polymerase during sequencing is referred to as a “polymerase read” or a Continuous Long Read (CLR). The polymerase runs through the template continuously, sequencing the DNA by adding nucleotides in both the forward and reverse orientation. Similar to the previous RSII platform, Pacbio Sequel uses the SMRTBell, a double stranded DNA molecule that loops around the ends, as the template for sequencing. Accuracy has also substantially improved from previous long-read platforms. In contrast to second-generation methodologies, PacBio provides longer length reads, in much less time, with greatly reduced-content bias, and an ability to distinguish between methylated and unmethylated bases. Recently introduced PacBio Sequel/Sequel2 platforms, which rely on Single-Molecule Real Time (SMRT) sequencing technology, are one of the most widely used long-read sequencing approaches. These new sequencing platforms are undergoing active development and pushing boundaries in terms of total output, read length, sequencing time, cost reduction and read accuracy. The primary contenders in third-generation sequencing are Pacific Biosciences (PacBio) (Sequel, Sequel2) and Oxford Nanopore (MinION, GridION, and PromethION). ![]() ![]() The third-generation of sequencing is here and making tremendous impact in the field of genomics. SequelTools is a program that provides the only free, fast, and easy-to-use quality control tool, and the only program providing this kind of read subsampling and read filtering for PacBio Sequel raw sequence data, and is available at. SequelTools is implemented in bash, R, and Python using only standard libraries and packages and is platform independent. The Read Filtering tool provides options for normalizing data by filtering out certain low-quality scraps reads and/or by minimum CLR length. ![]() The Read Subsampling tool allows the user to subsample reads by one or more of the following criteria: longest subreads per CLR or random CLR selection. ![]() The Quality Control tool quickly processes PacBio Sequel raw sequence data from multiple SMRTcells producing multiple statistics and publication-quality plots describing the quality of the data including N50, read length and count statistics, PSR, and ZOR. Here we present SequelTools, a command-line program containing three tools: Quality Control, Read Subsampling, and Read Filtering. Yet, hitherto no tool was available for analyzing the quality of, subsampling, and filtering PacBio data. PacBio sequencing is an incredibly valuable third-generation DNA sequencing method due to very long read lengths, ability to detect methylated bases, and its real-time sequencing methodology. ![]()
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