scholarly journals Nanopore sequencing provides rapid and reliable insight into microbial profiles of Intensive Care Units

2021 ◽  
Author(s):  
Guilherme Marcelino Viana de Siqueira ◽  
Felipe Marcelo Pereira-dos-Santos ◽  
Rafael Silva-Rocha ◽  
Maria-Eugenia Guazzaroni
Keyword(s):  
Intensive Care ◽  
Intensive Care Units ◽  
Nanopore Sequencing ◽  
Accurate Identification ◽  
Complex Samples ◽  
Healthcare Settings ◽  
Long Read ◽  
Single Use ◽  
Sequencing Platforms ◽  
Insight Into

Fast and accurate identification of pathogens is an essential task in healthcare settings. Next generation sequencing platforms such as Illumina have greatly expanded the capacity with which different organisms can be detected in hospital samples, and third-generation nanopore-driven sequencing devices such as Oxford Nanopore's minION have recently emerged as ideal sequencing platforms for routine healthcare surveillance due to their long-read capacity and high portability. Despite its great potential, protocols and analysis pipelines for nanopore sequencing are still being extensively validated. In this work, we assess the ability of nanopore sequencing to provide reliable community profiles based on 16S rRNA sequencing in comparison to traditional Illumina platforms using samples collected from Intensive Care Units from a hospital in Brazil. While our results point that lower throughputs may be a shortcoming of the method in more complex samples, we show that the use of single-use Flongle flowcells in nanopore sequencing runs can provide insightful information on the community composition in healthcare settings.

scholarly journals Nanopore Sequencing Provides Rapid and Reliable Insight Into Microbial Profiles of Intensive Care Units

2021 ◽  
Vol 9 ◽  
Author(s):  
Guilherme Marcelino Viana de Siqueira ◽  
Felipe Marcelo Pereira-dos-Santos ◽  
Rafael Silva-Rocha ◽  
María-Eugenia Guazzaroni
Keyword(s):  
Intensive Care ◽  
Intensive Care Units ◽  
Nanopore Sequencing ◽  
Accurate Identification ◽  
Complex Samples ◽  
Healthcare Settings ◽  
Long Read ◽  
Second Generation Sequencing ◽  
Single Use ◽  
Sequencing Platforms

Fast and accurate identification of pathogens is an essential task in healthcare settings. Second-generation sequencing platforms such as Illumina have greatly expanded the capacity with which different organisms can be detected in hospital samples, and third-generation nanopore-driven sequencing devices such as Oxford Nanopore's minION have recently emerged as ideal sequencing platforms for routine healthcare surveillance due to their long-read capacity and high portability. Despite its great potential, protocols and analysis pipelines for nanopore sequencing are still being extensively validated. In this work, we assess the ability of nanopore sequencing to provide reliable community profiles based on 16S rRNA sequencing in comparison to traditional Illumina platforms using samples collected from Intensive Care Units of a hospital in Brazil. While our results demonstrate that lower throughputs may be a shortcoming of the method in more complex samples, we show that the use of single-use Flongle flowcells in nanopore sequencing runs can provide insightful information on the community composition in healthcare settings.

scholarly journals Long-Read Sequencing of the Zebrafish Genome Reorganizes Genomic Architecture

2021 ◽  
Author(s):  
Yelena Chernyavskaya ◽  
Xiaofei Zhang ◽  
Jinze Liu ◽  
Jessica S. Blackburn
Keyword(s):  
Low Complexity ◽  
Zebrafish Genome ◽  
Nanopore Sequencing ◽  
Sequencing Technology ◽  
Short Read ◽  
Short Read Sequencing ◽  
Genomic Landscape ◽  
Long Reads ◽  
Long Read ◽  
Sequencing Platforms

Nanopore sequencing technology has revolutionized the field of genome biology with its ability to generate extra-long reads that can resolve regions of the genome that were previously inaccessible to short-read sequencing platforms. Although long-read sequencing has been used to resolve several vertebrate genomes, a nanopore-based zebrafish assembly has not yet been released. Over 50% of the zebrafish genome consists of difficult to map, highly repetitive, low complexity elements that pose inherent problems for short-read sequencers and assemblers. We used nanopore sequencing to improve upon and resolve the issues plaguing the current zebrafish reference assembly (GRCz11). Our long-read assembly improved the current resolution of the reference genome by identifying 1,697 novel insertions and deletions over 1Kb in length and placing 106 previously unlocalized scaffolds. We also discovered additional sites of retrotransposon integration previously unreported in GRCz11 and observed their expression in adult zebrafish under physiologic conditions, implying they have active mobility in the zebrafish genome and contribute to the ever-changing genomic landscape.

scholarly journals Highly accurate barcode and UMI error correction using dual nucleotide dimer blocks allows direct single-cell nanopore transcriptome sequencing

2021 ◽  
Author(s):  
Martin Philpott ◽  
Jonathan Watson ◽  
Anjan Thakurta ◽  
Tom Brown ◽  
Tom Brown ◽  
...  
Keyword(s):  
Single Cell ◽  
Nanopore Sequencing ◽  
Short Read ◽  
Short Read Sequencing ◽  
Single Cell Sequencing ◽  
Base Calling ◽  
Novel Approach ◽  
Long Read ◽  
First Time ◽  
Insight Into

AbstractDroplet-based single-cell sequencing techniques have provided unprecedented insight into cellular heterogeneities within tissues. However, these approaches only allow for the measurement of the distal parts of a transcript following short-read sequencing. Therefore, splicing and sequence diversity information is lost for the majority of the transcript. The application of long-read Nanopore sequencing to droplet-based methods is challenging because of the low base-calling accuracy currently associated with Nanopore sequencing. Although several approaches that use additional short-read sequencing to error-correct the barcode and UMI sequences have been developed, these techniques are limited by the requirement to sequence a library using both short- and long-read sequencing. Here we introduce a novel approach termed single-cell Barcode UMI Correction sequencing (scBUC-seq) to efficiently error-correct barcode and UMI oligonucleotide sequences synthesized by using blocks of dimeric nucleotides. The method can be applied to correct either short-read or long-read sequencing, thereby allowing users to recover more reads per cell and permits direct single-cell Nanopore sequencing for the first time. We illustrate our method by using species-mixing experiments to evaluate barcode assignment accuracy and evaluate differential isoform usage and fusion transcripts using myeloma and sarcoma cell line models.

scholarly journals Accurate profiling of forensic autosomal STRs using the Oxford Nanopore Technologies MinION device

2021 ◽  
Author(s):  
Courtney L. Hall ◽  
Rupesh K. Kesharwani ◽  
Nicole R. Phillips ◽  
John V. Planz ◽  
Fritz J. Sedlazeck ◽  
...  
Keyword(s):  
Specific Method ◽  
Nanopore Sequencing ◽  
Autosomal Strs ◽  
Str Typing ◽  
Str Loci ◽  
Oxford Nanopore ◽  
Long Read ◽  
Flanking Region ◽  
Sequencing Platforms ◽  
Oxford Nanopore Technologies

The high variability characteristic of short tandem repeat (STR) markers is harnessed for human identification in forensic genetic analyses. Despite the power and reliability of current typing techniques, sequence-level information both within and around STRs are masked in the length-based profiles generated. Forensic STR typing using next generation sequencing (NGS) has therefore gained attention as an alternative to traditional capillary electrophoresis (CE) approaches. In this proof-of-principle study, we evaluate the forensic applicability of the newest and smallest NGS platform available — the Oxford Nanopore Technologies (ONT) MinION device. Although nanopore sequencing on the handheld MinION offers numerous advantages, including on-site sample processing, the relatively high error rate and lack of forensic-specific analysis software has prevented accurate profiling across STR panels in previous studies. Here we present STRspy, a streamlined method capable of producing length- and sequence-based STR allele designations from noisy, long-read data. To demonstrate the capabilities of STRspy, seven reference samples (female: n = 2; male: n = 5) were amplified at 15 and 30 PCR cycles using the Promega PowerSeq 46GY System and sequenced on the ONT MinION device in triplicate. Basecalled reads were processed with STRspy using a custom database containing alleles reported in the STRSeq BioProject NIST 1036 dataset. Resultant STR allele designations and flanking region single nucleotide polymorphism (SNP) calls were compared to the manufacturer-validated genotypes for each sample. STRspy generated robust and reliable genotypes across all autosomal STR loci amplified with 30 PCR cycles, achieving 100% concordance based on both length and sequence. Furthermore, we were able to identify flanking region SNPs with >90% accuracy. These results demonstrate that nanopore sequencing platforms are capable of revealing additional variation in and around STR loci depending on read coverage. As the first long-read platform-specific method to successfully profile the entire panel of autosomal STRs amplified by a commercially available multiplex, STRspy significantly increases the feasibility of nanopore sequencing in forensic applications.

scholarly journals circFL-seq reveals full-length circular RNAs with rolling circular reverse transcription and nanopore sequencing

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Zelin Liu ◽  
Changyu Tao ◽  
Shiwei Li ◽  
Minghao Du ◽  
Yongtai Bai ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Large Scale ◽  
Full Length ◽  
Circular Rnas ◽  
Nanopore Sequencing ◽  
Accurate Identification ◽  
Functional Studies ◽  
Sequencing Method ◽  
Long Read ◽  
Fine Tune

Circular RNAs (circRNAs) act through multiple mechanisms via their sequence features to fine-tune gene expression networks. Due to overlapping sequences with linear cognates, identifying internal sequences of circRNAs remains a challenge, which hinders a comprehensive understanding of circRNA functions and mechanisms. Here, based on rolling circular reverse transcription (RCRT) and nanopore sequencing, we developed circFL-seq, a full-length circRNA sequencing method, to profile circRNA at the isoform level. With a customized computational pipeline to directly identify full-length sequences from rolling circular reads, we reconstructed 77,606 high-quality circRNAs from seven human cell lines and two human tissues. circFL-seq benefits from rolling circles and long-read sequencing, and the results showed more than tenfold enrichment of circRNA reads and advantages for both detection and quantification at the isoform level compared to those for short-read RNA sequencing. The concordance of the RT-qPCR and circFL-seq results for the identification of differential alternative splicing suggested wide application prospects for functional studies of internal variants in circRNAs. Moreover, the detection of fusion circRNAs at the omics scale may further expand the application of circFL-seq. Together, the accurate identification and quantification of full-length circRNAs make circFL-seq a potential tool for large-scale screening of functional circRNAs.

scholarly journals NanoSplicer: Accurate identification of splice junctions using Oxford Nanopore sequencing

2021 ◽  
Author(s):  
Yupei You ◽  
Michael B. Clark ◽  
Heejung Shim
Keyword(s):  
Cancer Cell Line ◽  
Splice Junction ◽  
Electrical Current ◽  
Lung Cancer Cell Line ◽  
Nanopore Sequencing ◽  
Lung Cancer Cell ◽  
Accurate Identification ◽  
Oxford Nanopore ◽  
Long Read ◽  
Splice Junctions

Motivation: Long read sequencing methods have considerable advantages for characterising RNA isoforms. Oxford nanopore sequencing records changes in electrical current when nucleic acid traverses through a pore. However, basecalling of this raw signal (known as a squiggle) is error prone, making it challenging to accurately identify splice junctions. Existing strategies include utilising matched short-read data and/or annotated splice junctions to correct nanopore reads but add expense or limit junctions to known (incomplete) annotations. Therefore, a method that could accurately identify splice junctions solely from nanopore data would have numerous advantages. Results: We developed "NanoSplicer" to identify splice junctions using raw nanopore signal (squiggles). For each splice junction the observed squiggle is compared to candidate squiggles representing potential junctions to identify the correct candidate. Measuring squiggle similarity enables us to compute the probability of each candidate junction and find the most likely one. We tested our method using 1. synthetic mRNAs with known splice junctions 2. biological mRNAs from a lung-cancer cell-line. The results from both datasets demonstrate NanoSplicer improves splice junction identification, especially when the basecalling error rate near the splice junction is elevated. Our method is implemented in the software package NanoSplicer, available at https://github.com/shimlab/NanoSplicer.

scholarly journals Identifying and correcting repeat-calling errors in nanopore sequencing of telomeres

2022 ◽  
Author(s):  
Kar-Tong Tan ◽  
Michael Slevin ◽  
Matthew Meyerson ◽  
Heng Li
Keyword(s):  
Genome Sequencing ◽  
Negative Impact ◽  
Electrical Current ◽  
Nanopore Sequencing ◽  
High Similarity ◽  
Potential Approach ◽  
Specific Manner ◽  
Long Read ◽  
Sequencing Platforms ◽  
Genomic Regions

Nanopore long-read genome sequencing is emerging as a potential approach for the study of genomes including long repetitive elements like telomeres. Here, we report extensive basecalling induced errors at telomere repeats across nanopore datasets, sequencing platforms, basecallers, and basecalling models. We found that telomeres which are represented by (TTAGGG)n and (CCCTAA)n repeats in many organisms were frequently miscalled (~40-50% of reads) as (TTAAAA)n, or as (CTTCTT)n and (CCCTGG)n repeats respectively in a strand-specific manner during nanopore sequencing. We showed that this miscalling is likely caused by the high similarity of current profiles between telomeric repeats and these repeat artefacts, leading to mis-assignment of electrical current profiles during basecalling. We further demonstrated that tuning of nanopore basecalling models, and selective application of the tuned models to telomeric reads led to improved recovery and analysis of telomeric regions, with little detected negative impact on basecalling of other genomic regions. Our study thus highlights the importance of verifying nanopore basecalls in long, repetitive, and poorly defined regions of the genome, and showcases how such artefacts in regions like telomeres can potentially be resolved by improvements in nanopore basecalling models.

scholarly journals circFL-seq reveals full-length circular RNAs with rolling circular reverse transcription and nanopore sequencing

2021 ◽  
Author(s):  
Zelin Liu ◽  
Changyu Tao ◽  
Shiwei Li ◽  
Minghao Du ◽  
Yongtai Bai ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Large Scale ◽  
Full Length ◽  
Circular Rnas ◽  
Nanopore Sequencing ◽  
Accurate Identification ◽  
Functional Studies ◽  
Sequencing Method ◽  
Long Read ◽  
Fine Tune

Circular RNAs (circRNAs) act through multiple mechanisms with their sequence features to fine-tune gene expression networks. Due to overlapping sequences with linear cognates, identifying internal sequences of circRNAs remains a great challenge, which hinders comprehensive understanding of circRNA functions and mechanisms. Here, based on rolling circular reverse transcription (RCRT) and nanopore sequencing, we developed circFL-seq, a full-length circRNA sequencing method, to profile circRNA at the isoform level. With a customized computational pipeline circfull to directly identify full-length sequences from rolling circular reads, we reconstructed 77,606 high-quality circRNAs from seven human cell lines and two human tissues. Benefiting from rolling circles and long-read sequencing, circFL-seq showed more than tenfold enrichment of circRNA reads and advantages for both detection and quantification at the isoform level compared to short-read RNA sequencing. The concordance of RT-qPCR and circFL-seq results for the identification of differential alternative splicing suggested wide application prospects for functional studies of internal variants in circRNAs. Moreover, the detection of cancer-related fusion circRNAs at the omics scale may further expand the application of circFL-seq. Together, the accurate identification and quantification of full-length circRNAs make circFL-seq a potential tool for large-scale screening of functional circRNAs.

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