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While 16S hypervariable region analysis is a powerful tool for bacterial taxonomic studies, it struggles to differentiate between closely related species. [35] In the families Enterobacteriaceae, Clostridiaceae, and Peptostreptococcaceae, species can share up to 99% sequence similarity across the full 16S gene. [37]
Sequence coverage (or depth) is the number of unique reads that include a given nucleotide in the reconstructed sequence. [1] [2] Deep sequencing refers to the general concept of aiming for high number of unique reads of each region of a sequence. [3] Physical coverage, the cumulative length of reads or read pairs expressed as a multiple of ...
Length Accession Reference; Bacterial (Prokaryotic) 16S: Escherichia coli: 1,541 nt J01859.1 [1] Archaeal (Prokaryotic) 16S Halobacterium salinarum: 1,473 nt
Sequencing by ligation (SOLiD sequencing) 50+35 or 50+50 bp: 99.9%: 1.2 to 1.4 billion: 1 to 2 weeks: $60–130: Low cost per base. Slower than other methods. Has issues sequencing palindromic sequences. [98] Nanopore Sequencing: Dependent on library preparation, not the device, so user chooses read length (up to 2,272,580 bp reported [99 ...
Sequencing platform choice and parameters are guided by experimental design and cost. Common experimental design considerations include deciding on the sequencing length, sequencing depth, use of single versus paired-end sequencing, number of replicates, multiplexing, randomization, and spike-ins. [19]
Rapid amplification of cDNA ends (RACE) is a technique used in molecular biology to obtain the full length sequence of an RNA transcript found within a cell. RACE results in the production of a cDNA copy of the RNA sequence of interest, produced through reverse transcription, followed by PCR amplification of the cDNA copies (see RT-PCR).
It can process single-end and paired-end sequencing data of arbitrary length. cutadapt [25] removes adapter sequences from next-generation sequencing data (Illumina, SOLiD and 454). It is used especially when the read length of the sequencing machine is longer than the sequenced molecule, like the microRNA case.
In bacteria and archaea, there is a single ITS, located between the 16S and 23S rRNA genes. Conversely, there are two ITSs in eukaryotes: ITS1 is located between 18S and 5.8S rRNA genes, while ITS2 is between 5.8S and 28S (in opisthokonts, or 25S in plants) rRNA genes. ITS1 corresponds to the ITS in bacteria and archaea, while ITS2 originated ...
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