Van Dongen, J. | Search bioRxiv

bioRxiv2024

Scywalker: scalable end-to-end data analysis workflow for nanopore single-cell transcriptome sequencing

We introduce scywalker, an innovative and scalable package developed to comprehensively analyze long-read nanopore sequencing data of full-length single-cell or single-nuclei cDNA. Existing nanopore single-cell data analysis tools showed severe limitations in handling current data sizes. We developed novel scalable methods for cell barcode demultiplexing and single-cell isoform calling and quantification and incorporated these in an easily deployable package. Scywalker streamlines the entire analysis process, from sequenced fragments in FASTQ format to demultiplexed pseudobulk isoform counts, into a single command suitable for execution on either server or cluster. Scywalker includes data quality control, cell type identification, and an interactive report. Assessment of datasets from the human brain, Arabidopsis leaves, and previously benchmarked data from mixed cell lines, demonstrate excellent correlation with short-read analyses at both the cell-barcoding and gene quantification levels. At the isoform level, we show that scywalker facilitates the direct identification of cell-type-specific expression of novel isoforms.

bioinformatics↗

bioRxiv2018

Accurate characterization of expanded tandem repeat length and sequence through whole genome long-read sequencing on PromethION.

Tandem repeats (TRs) can cause disease through their length, sequence motif interruptions, and nucleotide modifications. For many TRs, however, these features are very difficult - if not impossible - to assess, requiring low-throughput and labor-intensive assays. One example is a VNTR in ABCA7 for which we recently discovered that expanded alleles strongly increase risk of Alzheimers disease. Here, we investigated the potential of long-read whole genome sequencing to surmount these challenges, using the high-throughput PromethION platform from Oxford Nanopore Technologies. To overcome the limitations of conventional base calling and alignment, we developed an algorithm to study the TR size and sequence directly on raw PromethION current data.\n\nWe report the long-read sequencing of multiple human genomes (n = 11) using only a single sequencing run and flow cell per individual. With the use of fresh DNA extractions, DNA shearing to approximately 20kb and size selection, we obtained an average output of 70 gigabases (Gb) per flow cell, corresponding to a 21x genome coverage, and a maximum yield of 98 Gb (30x genome coverage). All ABCA7 VNTR alleles, including expansions up to 10,000 bases, were spanned by long sequencing reads, validated by Southern blotting. Classical approaches of TR length estimation suffered from low accuracy, low precision, DNA strand effects and/or inability to call pathogenic repeat expansions. In contrast, our novel NanoSatellite algorithm, which circumvents base calling by using dynamic time warping on raw PromethION current data, achieved more than 90% accuracy and high precision (5.6% relative standard deviation) of TR length estimation, and detected all clinically relevant repeat expansions. In addition, we identified alternative TR sequence motifs with high consistency, allowing determination of TR sequence and distinction of VNTR alleles with homozygous length.\n\nIn conclusion, we validated the robustness of single-experiment whole genome long-read sequencing on PromethION, a prerequisite for application of long-read sequencing in the clinic. In addition, we outperformed Southern blotting, enabling improved characterization of the role of expanded ABCA7 VNTR alleles in Alzheimers disease, and opening new opportunities for TR research.

genomics↗