ArticleMolecular biology reports2023
Optimization of long-range PCR protocol to prepare filaggrin exon 3 libraries for PacBio long-read sequencing.
Article in Molecular biology reports, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 6 papers.
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Who cites it
6 citing papers in PubMed, 7 citations in OpenAlex.
- High-Quality Genome Assemblies of Two Prototheca wickerhamii Strains.Scientific data · 2026Article
- Novel loss-of-function variants in filaggrin exon 3 in patients with severe atopic dermatitis.Archives of dermatological research · 2024Article
- Filaggrin loss-of-function variants are associated with atopic dermatitis phenotypes in a diverse, early-life prospective cohort.JCI insight · 2024Article
- Characterization and genome-informatic analysis of a novel lytic mendocina phage vB_PmeS_STP12 suitable for phage therapy pseudomonas or biocontrol.Molecular biology reports · 2024Article
- Towards a Long-Read Sequencing Approach for the Molecular Diagnosis of RPGRInternational journal of molecular sciences · 2023Article
- Improving Yields in Multi-analyte Extractions by Utilizing Post-homogenized Tissue Debris.The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society · 2023Article
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Authors and funding
12 authors at 3 institutions in 1 country.
Funding
Abstract
backgroundThe filaggrin (FLG) protein, encoded by the FLG gene, is an intermediate filament-associated protein that plays a crucial role in the terminal stages of human epidermal differentiation. Loss-of-function mutations in the FLG exon 3 have been associated with skin diseases. The identification of causative mutations is challenging, due to the high sequence homology within its exon 3 (12,753 bp), which includes 10 to 12 filaggrin tandem repeats. With this study we aimed to obtain the whole FLG exon 3 sequence through PacBio technology, once 13-kb amplicons have been generated. METHODS AND
resultsFor the preparation of SMRTbell libraries to be sequenced using PacBio technology, we focused on optimizing a 2-step long-range PCR protocol to generate 13-kb amplicons covering the whole FLG exon 3 sequence. The performance of three long-range DNA polymerases was assessed in an attempt to improve the PCR conditions required for the enzymes to function properly. We focused on optimization of the input template DNA concentration and thermocycling parameters to correctly amplify the entire FLG exon 3 sequence, minimizing non-specific amplification.
conclusionsTaken together, our findings suggested that the PrimeSTAR protocol is suitable for producing the amplicons of the 13-kb FLG whole exon 3 to prepare SMRTbell libraries. We suggest that sequencing the generated amplicons may be useful for identifying LoF variants that are causative of the patients' disorders.
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