Chapter 53 : Practices of Sequencing Quality Assurance

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While the breakthrough discovery of DNA structure was in 1953, many innovations still needed to occur before labs could generate the sequential order of nucleotides in genetic material. Results began to materialize 15 years later in 1968, when Wu and Kaiser performed “cohesive end” sequencing on phage DNA using radiolabeled nucleotides and DNA polymerase (1). This method produced reads of 10 to 20 bases, and fragments were separated by two-dimensional chromatography.

Citation: Norman K, Dinauer D. 2016. Practices of Sequencing Quality Assurance, p 766-783. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch53
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Image of FIGURE 1

(A) Sanger sequencing workflow. (B) Terminator nucleotides prevent further polymerization and label each fragment with a dye corresponding to the final nucleotide identity.

Citation: Norman K, Dinauer D. 2016. Practices of Sequencing Quality Assurance, p 766-783. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch53
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Image of FIGURE 2

Next-generation sequencing workflow. See text for details.

Citation: Norman K, Dinauer D. 2016. Practices of Sequencing Quality Assurance, p 766-783. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch53
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Image of FIGURE 3

(A) Direct PCR. (B) Fusion PCR library preparation strategies. (C) Mate-paired reads are separated by a known intervening sequence length (dashed lines), allowing for accurate relative positional alignment. See text for details.

Citation: Norman K, Dinauer D. 2016. Practices of Sequencing Quality Assurance, p 766-783. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch53
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Image of FIGURE 4

Image of mixed base traces viewed in RECall software showing examples of mixed base variation in HIV Sanger assay at two codon positions: amino acids 37 (aa37) and 41 (aa41). Each panel consists of sequencing traces from the forward sequencing primer (PRT-F2) and the reverse sequencing primer (AV44). Mixed base positions are identified by the software and labeled with IUB codes: Y = T + C, R = A + G, W = A + T, H = C + T. The RECall software aligns all sequences to a reference at the top of each panel and shows the composite base call below the reference. The highlighted T position across all the panels is a vertical alignment position between the panels. Panel A shows 2 codons (37 and 41 of RT) with nucleotide base-calling of AYR; panel B shows the AWR at codon 41 (the second peaks at codon 37 were not detected in this replicate); panel C shows ACR at codon 37 (minor T was not called by RECall at the cutoff of 15%) and AHR at codon 41 (almost equal height of second and third peak at the second position) ( ).

Citation: Norman K, Dinauer D. 2016. Practices of Sequencing Quality Assurance, p 766-783. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch53
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Examples of sequencing technology applications

Citation: Norman K, Dinauer D. 2016. Practices of Sequencing Quality Assurance, p 766-783. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch53
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Selected resources for sequencing assay development

Citation: Norman K, Dinauer D. 2016. Practices of Sequencing Quality Assurance, p 766-783. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch53
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Examples of analytical validation strategies

Citation: Norman K, Dinauer D. 2016. Practices of Sequencing Quality Assurance, p 766-783. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch53
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Example nucleic acid input amounts across various platforms

Citation: Norman K, Dinauer D. 2016. Practices of Sequencing Quality Assurance, p 766-783. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch53

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