Facilitated Large-Scale Sequence Validation Platform Using Tn5-Tagmented Cell Lysates

Hwang, Byung Kook; Heo, Sunghoon; Cho, Namjin; Seo, Han-Na; Bang, Duhee

doi:10.1021/acssynbio.8b00482

Cited by 8 publications

(2 citation statements)

References 6 publications

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“…Analogous to colony PCR, where lysed bacterial cells or yeast spheroplasts are added to a PCR reaction without prior genomic DNA isolation, we hypothesized that yeast whole-genome sequencing library preparation could be simplified by applying tagmentation directly to cells. A similar strategy incorporating heat-based lysis prior to tagmentation was successfully applied for whole-genome 1 and plasmid 7 sequencing of bacterial cells. In contrast to bacteria, the yeast S. cerevisiae has a rigid cell wall that prevents efficient heatbased lysis.…”

Section: Introductionmentioning

confidence: 99%

Fast and inexpensive whole genome sequencing library preparation from intact yeast cells

Vonesch

Szu-Tu

et al. 2020

Preprint

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Through the increase in the capacity of sequencing machines massively parallel sequencing of thousands of samples in a single run is now possible. With the improved throughput and resulting drop in the price of sequencing, the cost and time for preparation of sequencing libraries have become the major bottleneck in large-scale experiments. Methods using a hyperactive variant of the Tn5 transposase efficiently generate libraries starting from cDNA or genomic DNA in a few hours and are highly scalable. For genome sequencing, however, the time and effort spent on genomic DNA isolation limits the practicability of sequencing large numbers of samples. Here, we describe a highly scalable method for preparing high quality whole-genome sequencing libraries directly from yeast cultures in less than three hours at 34 cents per sample. We skip the rate-limiting step of genomic DNA extraction by directly tagmenting yeast spheroplasts and add a nucleosome release step prior to enrichment PCR to improve the evenness of genomic coverage. Resulting libraries do not show any GC-bias and are comparable in quality to libraries processed from genomic DNA with a commercially available Tn5-based kit. We use our protocol to investigate CRISPR/Cas9 on- and off-target edits and reliably detect edited variants and shared polymorphisms between strains. Our protocol enables rapid preparation of unbiased and high-quality, sequencing-ready indexed libraries for hundreds of yeast strains in a single day at a low price. By adjusting individual steps of our workflow we expect that our protocol can be adapted to other organisms.

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Section: Introductionmentioning

confidence: 99%

Fast and inexpensive whole genome sequencing library preparation from intact yeast cells

Vonesch

Szu-Tu

et al. 2020

Preprint

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“…Subsequently, conventional RT-PCR was performed with published primers to amplify the RV gene segments [7] with the additional VP4 forward primer VP4_P9_F1 (5′-GGC TAT AAA ATG GCT TCT TTAAT-3′) and reverse primer VP4_P9_R2359 (5′-GGT CAC ATC TTA AAA TAG ACAG-3′). Next, amplicons were subjected to barcode-tagged next-generation sequencing (Celemics, Seoul, Korea) [8]. Nucleotide sequence output representing the nearly complete individual gene segments and their corresponding chromatograms were analyzed using BioEdit [9].…”

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confidence: 99%

A G3P[9] rotavirus strain with an unusual genome constellation in a diarrheic cat in Thailand

Lestari

Chandranoi²,

Chuchaona

et al. 2023

Arch Virol

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Rotavirus infection can cause diarrhea in many animal species. A 2-year-old indoor female Siamese cat with bloody mucoid diarrhea tested positive for rotavirus (RV) group A by real-time reverse transcription polymerase chain reaction (RT-PCR). Subsequent conventional RT-PCR amplification of the 11 RV segments and sequencing revealed a G3-P[9]-I2-R2-C2-M2-A3-N2-T3-E3-H3 genome constellation. Phylogenetic analysis showed that the VP4, VP7, NSP1, NSP3, NSP4, and NSP5 genes were closely related to those of human feline-like rotaviruses, while the VP1, VP2, VP3, VP6, and NSP2 genes were genetically closest to those of human bovine-like rotaviruses, suggesting that genetic reassortment had occurred. The uniqueness of this G3P[9] feline rotavirus strain expands our knowledge about feline rotaviruses. Supplementary Information The online version contains supplementary material available at 10.1007/s00705-022-05641-1.

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Natural infection of Cnidoscolus and Jatropha by Sri Lankan cassava mosaic virus in Thailand

Hemniam

Roekwan²,

Vannatim³

et al. 2022

J Gen Plant Pathol

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Facilitated Large-Scale Sequence Validation Platform Using Tn5-Tagmented Cell Lysates

Cited by 8 publications

References 6 publications

Fast and inexpensive whole genome sequencing library preparation from intact yeast cells

Fast and inexpensive whole genome sequencing library preparation from intact yeast cells

A G3P[9] rotavirus strain with an unusual genome constellation in a diarrheic cat in Thailand

Natural infection of Cnidoscolus and Jatropha by Sri Lankan cassava mosaic virus in Thailand

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