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Peccoud, S. J.

Publications and source records attributed to Peccoud, S. J..

2 recordsLinked to original sources

Self-Documenting Plasmids

SO_SCPLOWUMMARYC_SCPLOWPlasmids are the workhorse of biotechnology. These small DNA molecules are used to produce recombinant proteins and to engineer living organisms. They can be regarded as the blueprints of many biotechnology products. It is, therefore, critical to ensure that the sequences of these DNA molecules match their intended designs. Yet, plasmid verification remains challenging. To secure the exchange of plasmids in research and development workflows, we have developed self-documenting plasmids that encode information about themselves in their own DNA molecules. Users of self-documenting plasmids can retrieve critical information about the plasmid without prior knowledge of the plasmid identity. The insertion of documentation in the plasmid sequence does not adversely affect their propagation in bacteria and does not compromise protein expression in mammalian cells. This technology simplifies plasmid verification, hardens supply chains, and has the potential to transform the protection of intellectual property in the life sciences.

synthetic biology↗

Hybrid Sequencing Facilitates Robust De Novo Plasmid Assembly

Despite the wide use of plasmids in research and clinical production, the need to verify plasmid sequences is a bottleneck that is too often underestimated in the manufacturing process. Although sequencing platforms continue to improve, the method and assembly pipeline chosen still influence the final plasmid assembly sequence. Furthermore, few dedicated tools exist for plasmid assembly, especially for de novo assembly. Here, we evaluated short-read, long-read, and hybrid (both short and long reads) de novo assembly pipelines across three replicates of a 24-plasmid library. Consistent with previous characterizations of each sequencing technology, short-read assemblies had issues resolving GC-rich regions, and long-read assemblies commonly had small insertions and deletions, especially in repetitive regions. The hybrid approach facilitated the most accurate, consistent assembly generation and identified mutations relative to the reference sequence. Although Sanger sequencing can be used to verify specific regions, some GC-rich and repetitive regions were difficult to resolve using any method, suggesting that easily sequenced genetic parts should be prioritized in the design of new genetic constructs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/586694v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@13945c1org.highwire.dtl.DTLVardef@11287a8org.highwire.dtl.DTLVardef@1885654org.highwire.dtl.DTLVardef@1dbdbf3_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG

synthetic biology↗