© F e r r a t a S t o r t i F o u n d a t i o nregions and flanking intronic regions of VWF (~10 Kb) are included ( Figure 1A). Normalization of amplicons was carried out by gel quantification performed with ImageJ (Version 1.43u), a public domain Java image processing program (http://rsbweb.nih.gov/ij/). By taking a sample with a known concentration as the control, it was possible to extrapolate the concentration of each sample to create a normalized pool of the 47 PCR products in one tube. This pooling process produced 8 tubes and each tube was the result of simultaneous amplification of VWF of 5 patients. Polymerase chain reaction fragmentation, massively parallel sequencing and bioinformatic analysisIn NGS, the fragmentation step is critical for the success of the sequencing process and must be performed according to stringent parameters. Because the commonly used mechanical systems for genomic DNA fragmentation such as sonication are not effective for short DNA fragments (e.g., <300-500 bp), 12 conventional short PCRs were fragmented by heat incubation at 95ºC for 9 h ( Figure 1B) followed by 1 h of re-annealing at 72°C. This provided efficient DNA fragmentation that was relatively unbiased, as required for Illumina GA sequencing procedures.12 All samples were purified using the QIAquick PCR Purification Kit (Qiagen) and the size distribution of the fragments was checked using a DNA 100 chip on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Subsequent massively parallel sequencing and bioinformatic analysis are described in the Online Supplementary Design and Methods section. Mutation assignmentTo confirm the mutations identified by NGS and assign them to patients, the specific region including the mutation was PCRamplified and sequenced by dideoxynucleotide method, as described. 4 The sequences obtained were assembled and aligned against the consensus wild-type VWF sequence using SeqScape software (v2.7) (Applied Biosystems, Foster City, CA, USA). Results and DiscussionBased on the conclusions from a pilot study (Online Supplementary Appendix), a proof-of-concept study was performed for the simultaneous analysis by NGS of 40 VWD patients previously diagnosed and classified according to their clinical characteristics (Table 1). The mutations responsible for the disease were known in 8 of the selected patients who had been characterized by Sanger sequencing 4 and were used as controls. Patients included in this study were randomly selected from all those who met the criteria for VWD, to obtain a representative picture of the types, percentages, and variability of patients usually seen at the Hemophilia Unit.NGS technologies are economically extremely advantageous when the sequencer runs completely full which, in the case of short region sequencing (e.g., VWF), means including the maximum number of samples 14 and the preparation of one sequencing library per sample. However, library construction is one of the most expensive steps in the overall NGS process. Therefore, when t...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.