1. Single-Molecule Sequencing: Towards Clinical Applications
Adam Ameur, Wigard P. Kloosterman, Matthew S. Hestand 
Trends in Biotechnology 
Volume 37, Issue 1, Pages (January 2019)
DOI: /j.tibtech
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2. Figure 1 Key Figure: Key Advantages of Single-Molecule Sequencing
(A) PacBio can generate read length N50 values of around 20kb, while some specialized protocols for ONT can give N50 values of up to 100kb or more. This is much longer that what can be obtained for Sanger sequencing or short-read NGS. (B) Base modifications in a DNA molecule (represented by a brown hexagon) give rise to specific signals in the single-molecule sequencing data that can be identified using computational methods (represented by brown asterisks). (C) A PacBio library is a circular molecule composed of both strands of DNA (red and purple lines) connected by adaptors (black loops) (1). As a polymerase (circle with halo) copies the templates, this gives rise to several independent sub-reads (blue and orange lines), from each strand of the original molecule (2). Each sub-read contains a number of sequencing errors (red X’s), randomly distributed over the molecules. Sub-reads can then be computationally combined into a highly accurate circular consensus sequence (3). (D) Some ONT platforms are small enough to be conveniently carried around making them ideal for field studies. The picture shows a handheld MinION sequencer.
Trends in Biotechnology  , 72-85DOI: ( /j.tibtech )
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3. Figure 2
Tandem Repeat Sequencing Example: The Fragile X Loci. Ten PacBio CCS reads from female patient 21 in a study by Ardui and colleagues [21] trimmed to ±10bp around the FMR1 CGG repeat. Reads 0–4 come from the patient’s normal allele, whereas reads 5–9 come from the patient’s longer premutation allele. Both also contain AGG interruptions, indicated by arrows, which influence repeat stability in future generations.
Trends in Biotechnology  , 72-85DOI: ( /j.tibtech )
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4. Figure 3
Detection of Structural Variations by Long-Read Sequencing. (A) Pie charts indicating the number of novel PacBio insertions and deletions discovered by Chaisson and colleagues [37] in the human genome compared to known variants, as well as the amount of bases impacted by these variants. (B) Schematic illustrating assembly of long reads to identify (disease-related) structural variations. (C) Illustration of the use of direct mapping of long reads to a reference genome to resolve complex structural variations.
Trends in Biotechnology  , 72-85DOI: ( /j.tibtech )
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5. Figure 4
Genome Assemblies by Sequencing Technology. N50s of genome assemblies demonstrate long read-based assemblies greatly exceed NGS platform assemblies and even near human reference genome (hg38) N50 (indicated by a broken line). Data points are contig N50 versus coverage for all non-partial de novo human assemblies at National Center for Biotechnology Information ( from 2010 to July 2017 (only if annotated for both statistics) and the ONT assemblies from [64].
Trends in Biotechnology  , 72-85DOI: ( /j.tibtech )
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6. Figure 5
Clonal Evolution of Cancer Mutations during Treatment. A patient diagnosed with lower-risk myelodysplastic syndrome was tested for mutations in TP53 by Lodé and colleagues [70] at two distinct time points (August 2008 and July 2013) during the course of treatment. (A) Allele frequency of mutated TP53 molecules. The lowest frequency TP53 mutations (red lines) were only detected by PacBio sequencing, while the more common mutations were detected also by short-read NGS (Illumina sequencing) (see blue lines). The TP53 molecule carrying the mutation c.818G>A [marked by one asterisk (*)] was only detected by PacBio and not by Illumina at the first time point (August 2008) but was detected by both systems in sample 2 (July 2013). The TP53 molecule represented by two asterisks (**) was detected by PacBio in sample 1 but fell under the detection limit in sample 2. (B) Graphic representation of all distinct molecules with TP53 mutations in sample 2. PacBio sequencing was performed on a 3.6-kb amplicon encompassing six exons of TP53, and a phasing analysis showed that there were eight distinct TP53 molecules present in the sample, each containing one single mutation. The PacBio phasing reveals information about the clonal evolution of cancer mutations that is not possible to obtain using short-read NGS.
Trends in Biotechnology  , 72-85DOI: ( /j.tibtech )
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