A novel use of random priming-based single-strand library preparation for whole genome sequencing of formalin-fixed paraffin-embedded tissue samples

Saunderson, Emily A.; Baker, Ann‐Marie; Williams, Marc; Curtius, Kit; Jones, J. Louise; Graham, Trevor A.; Ficz, Gabriella

doi:10.1093/nargab/lqz017

Cited by 4 publications

(2 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Metabarcoding approaches paired with NGS to determine the species represented in mixed biological samples offers a tantalizing opportunity to mine vast amounts of sequence data from individual samples, including identification of pathogens, hosts, and vector from a single NGS run [ 187 ]. Unlike traditional PCR where primers must be designed in advance, NGS can use random primers during preparation of cDNA that allow for sampling of the true diversity of arthropod bloodmeals because the amplification is not biased by primer design [ 126 , 127 ]. Even when specific primers are used, the depth of coverage and accuracy offered by this technology can often differentiate the sequences of nuclear mitochondrial DNA (NUMTs) from mtDNA, clarifying species identification for mtDNA targets [ 128 ].…”

Section: Molecular Advancesmentioning

confidence: 99%

Modernizing the Toolkit for Arthropod Bloodmeal Identification

Borland

Kading

2021

Insects

View full text Add to dashboard Cite

Understanding vertebrate–vector interactions is vitally important for understanding the transmission dynamics of arthropod-vectored pathogens and depends on the ability to accurately identify the vertebrate source of blood-engorged arthropods in field collections using molecular methods. A decade ago, molecular techniques being applied to arthropod blood meal identification were thoroughly reviewed, but there have been significant advancements in the techniques and technologies available since that time. This review highlights the available diagnostic markers in mitochondrial and nuclear DNA and discusses their benefits and shortcomings for use in molecular identification assays. Advances in real-time PCR, high resolution melting analysis, digital PCR, next generation sequencing, microsphere assays, mass spectrometry, and stable isotope analysis each offer novel approaches and advantages to bloodmeal analysis that have gained traction in the field. New, field-forward technologies and platforms have also come into use that offer promising solutions for point-of-care and remote field deployment for rapid bloodmeal source identification. Some of the lessons learned over the last decade, particularly in the fields of DNA barcoding and sequence analysis, are discussed. Though many advancements have been made, technical challenges remain concerning the prevention of sample degradation both by the arthropod before the sample has been obtained and during storage. This review provides a roadmap and guide for those considering modern techniques for arthropod bloodmeal identification and reviews how advances in molecular technology over the past decade have been applied in this unique biomedical context.

show abstract

Section: Molecular Advancesmentioning

confidence: 99%

Modernizing the Toolkit for Arthropod Bloodmeal Identification

Borland

Kading

2021

Insects

View full text Add to dashboard Cite

show abstract

“…Therefore, they constitute an extensive repository of tissue material usable for long-term clinical diagnostics [ 22 ]. However, the use of FFPE tissues had several technical challenges, such as high content of human genomic DNA, low bacterial DNA [ 22 ], and sequence artifacts in the DNA extracted [ 23 ].…”

Section: Introductionmentioning

confidence: 99%

Colorectal Cancer Archaeome: A Metagenomic Exploration, Tunisia

Mathlouthi,

Oumarou Hama,

Belguith

et al. 2023

CIMB

View full text Add to dashboard Cite

Colorectal cancer (CRC) is a serious public health problem known to have a multifactorial etiology. The association between gut microbiota and CRC has been widely studied; however, the link between archaea and CRC has not been sufficiently studied. To investigate the involvement of archaea in colorectal carcinogenesis, we performed a metagenomic analysis of 68 formalin-embedded paraffin fixed tissues from tumoral (n = 33) and healthy mucosa (n = 35) collected from 35 CRC Tunisian patients. We used two DNA extraction methods: Generead DNA FFPE kit (Qiagen, Germantown, MD, USA) and Chelex. We then sequenced the samples using Illumina Miseq. Interestingly, DNA extraction exclusively using Chelex generated enough DNA for sequencing of all samples. After data filtering and processing, we reported the presence of archaeal sequences, which represented 0.33% of all the reads generated. In terms of abundance, we highlighted a depletion in methanogens and an enrichment in Halobacteria in the tumor tissues, while the correlation analysis revealed a significant association between the Halobacteria and the tumor mucosa (p < 0.05). We reported a strong correlation between Natrialba magadii, Sulfolobus acidocaldarius, and tumor tissues, and a weak correlation between Methanococcus voltae and healthy adjacent mucosa. Here, we demonstrated the feasibility of archaeome analysis from formol fixed paraffin-embedded (FFPE) tissues using simple protocols ranging from sampling to data analysis, and reported a significant association between Halobacteria and tumor tissues in Tunisian patients with CRC. The importance of our study is that it represents the first metagenomic analysis of Tunisian CRC patients’ gut microbiome, which consists of sequencing DNA extracted from paired tumor-adjacent FFPE tissues collected from CRC patients. The detection of archaeal sequences in our samples confirms the feasibility of carrying out an archaeome analysis from FFPE tissues using a simple DNA extraction protocol. Our analysis revealed the enrichment of Halobacteria, especially Natrialba magadii, in tumor mucosa compared to the normal mucosa in CRC Tunisian patients. Other species were also associated with CRC, including Sulfolobus acidocaldarius and Methanococcus voltae, which is a methanogenic archaea; both species were found to be correlated with adjacent healthy tissues.

show abstract

An efficient procedure for the recovery of DNA from formalin-fixed paraffin-embedded tissue sections

Oba

Kohashi

Sangatsuda

et al. 2022

Biology Methods and Protocols

View full text Add to dashboard Cite

With the advent of new molecular diagnostic techniques, retrieving DNA from the formalin-fixed paraffin-embedded (FFPE) tissues has become an essential yet challenging step for efficient downstream processes. Owing to low quality and quantity of DNA retrieved from the FFPE sections, the process is often impractical and needs significant improvements. Here, we established an efficient method for the purification of DNA from FFPE specimens by optimizing incubation temperature, incubation time, and the concentration of a formalin scavenger tris(hydroxymethyl)aminomethane (Tris) for reverse-crosslinking. The optimized method, named "Highly concentrated Tris-mediated DNA extraction" (HiTE), yielded three times the DNA yield per tissue slice compared with a representative DNA extraction kit. Moreover, the use of HiTE-extracted DNA increased the yield of the sequencing library three times and accordingly yielded a log higher and more reproducible sequencing library compared to that obtained using the commonly used commercial kit. The sequencing library prepared from HiTE-extracted FFPE-DNA had longer inserts and produced reads that evenly covered the reference genome. Successful application of HiTE-extracted FFPE-DNA for whole-genome and targeted gene panel sequencing indicates its practical usability.

show abstract

A novel use of random priming-based single-strand library preparation for whole genome sequencing of formalin-fixed paraffin-embedded tissue samples

Cited by 4 publications

References 20 publications

Modernizing the Toolkit for Arthropod Bloodmeal Identification

Modernizing the Toolkit for Arthropod Bloodmeal Identification

Colorectal Cancer Archaeome: A Metagenomic Exploration, Tunisia

An efficient procedure for the recovery of DNA from formalin-fixed paraffin-embedded tissue sections

Contact Info

Product

Resources

About