Multi-pass, single-molecule nanopore reading of long protein strands with single-amino acid sensitivity.

The ability to sequence single protein molecules in their native, full-length form would enable a more comprehensive understanding of proteomic diversity. Current technologies, however, are limited in achieving this goal. Here, we establish a method for long-range, single-molecule reading of intact protein strands on a commercial nanopore sensor array. By using the ClpX unfoldase to ratchet proteins through a CsgG nanopore, we achieve single-amino acid level sensitivity, enabling sequencing of combinations of amino acid substitutions across long protein strands. For greater sequencing accuracy, we demonstrate the ability to reread individual protein molecules, spanning hundreds of amino acids in length, multiple times, and explore the potential for high accuracy protein barcode sequencing. Further, we develop a biophysical model that can simulate raw nanopore signals a priori, based on amino acid volume and charge, enhancing the interpretation of raw signal data. Finally, we apply these methods to examine intact, folded protein domains for complete end-to-end analysis. These results provide proof-of-concept for a platform that has the potential to identify and characterize full-length proteoforms at single-molecule resolution.

Benefits, challenges, and usability evaluation of DeloreanJS: a back-in-time debugger for JavaScript.

JavaScript Web applications are a common product in industry. As with most applications, Web applications can acquire software flaws (known as bugs), whose symptoms are seen during the development stage and, even worse, in production. The use of debuggers is beneficial for detecting bugs. Unfortunately, most JavaScript debuggers (1) only support the "step into/through" feature in an execution program to detect a bug, and (2) do not allow developers to go back-in-time at the application execution to take actions to detect the bug accurately. For example, the second limitation does not allow developers to modify the value of a variable to fix a bug while the application is running or test if the same bug is triggered with other values of that variable. Using concepts such as continuations and static analysis, this article presents a usable debugger for JavaScript, named DeloreanJS, which enables developers to go back-in-time in different execution points and resume the execution of a Web application to improve the understanding of a bug, or even experiment with hypothetical scenarios around the bug. Using an online and available version, we illustrate the benefits of DeloreanJS through five examples of bugs in JavaScript. Although DeloreanJS is developed for JavaScript, a dynamic prototype-based object model with side effects (mutable variables), we discuss our proposal with the state-of-art/practice of debuggers in terms of features. For example, modern browsers like Mozilla Firefox include a debugger in their distribution that only support for the breakpoint feature. However DeloreanJS uses a graphical user interface that considers back-in-time features. The aim of this study is to evaluate and compare the usability of DeloreanJS and Mozilla Firefox's debugger using the system usability scale approach. We requested 30 undergraduate students from two computer science programs to solve five tasks. Among the findings, we highlight two results. First, we found that 100% (15) of participants recommended DeloreanJS, and only 53% (eight) recommended Firefox's debugger to complete the tasks. Second, whereas the average score for DeloreanJS is 71.6 ("Good"), the average score for Firefox's debugger is 55.8 ("Acceptable").

NGSEP 4: Efficient and accurate identification of orthogroups and whole-genome alignment.

Whole-genome alignment allows researchers to understand the genomic structure and variation among genomes. Approaches based on direct pairwise comparisons of DNA sequences require large computational capacities. As a consequence, pipelines combining tools for orthologous gene identification and synteny have been developed. In this manuscript, we present the latest functionalities implemented in NGSEP 4, to identify orthogroups and perform whole genome alignments. NGSEP implements functionalities for identification of clusters of homologus genes, synteny analysis and whole genome alignment. Our results showed that the NGSEP algorithm for orthogroups identification has competitive accuracy and efficiency in comparison to commonly used tools. The implementation also includes a visualization of the whole genome alignment based on synteny of the orthogroups that were identified, and a reconstruction of the pangenome based on frequencies of the orthogroups among the genomes. NGSEP 4 also includes a new graphical user interface based on the JavaFX technology. We expect that these new developments will be very useful for several studies in evolutionary biology and population genomics.

Multiplexed direct detection of barcoded protein reporters on a nanopore array.

Detection of specific proteins using nanopores is currently challenging. To address this challenge, we developed a collection of over twenty nanopore-addressable protein tags engineered as reporters (NanoporeTERs, or NTERs). NTERs are constructed with a secretion tag, folded domain and a nanopore-targeting C-terminal tail in which arbitrary peptide barcodes can be encoded. We demonstrate simultaneous detection of up to nine NTERs expressed in bacterial or human cells using MinION nanopore sensor arrays.

Herding cats: Label-based approaches in protein translocation through nanopore sensors for single-molecule protein sequence analysis.

Proteins carry out life's essential functions. Comprehensive proteome analysis technologies are thus required for a full understanding of the operating principles of biological systems. While current proteomics techniques suffer from limitations in sensitivity and/or throughput, nanopore technology has the potential to enable de novo protein identification through single-molecule sequencing. However, a significant barrier to achieving this goal is controlling protein/peptide translocation through the nanopore sensor for processive strand analysis. Here, we review recent approaches that use a range of techniques, from oligonucleotide conjugation to molecular motors, aimed at driving protein strands and peptides through protein nanopores. We further discuss site-specific protein conjugation chemistry that could be combined with these translocation approaches as future directions to achieve single-molecule protein detection and sequencing of native proteins.

NGSEP3: accurate variant calling across species and sequencing protocols.

MOTIVATION: Accurate detection, genotyping and downstream analysis of genomic variants from high-throughput sequencing data are fundamental features in modern production pipelines for genetic-based diagnosis in medicine or genomic selection in plant and animal breeding. Our research group maintains the Next-Generation Sequencing Experience Platform (NGSEP) as a precise, efficient and easy-to-use software solution for these features. RESULTS: Understanding that incorrect alignments around short tandem repeats are an important source of genotyping errors, we implemented in NGSEP new algorithms for realignment and haplotype clustering of reads spanning indels and short tandem repeats. We performed extensive benchmark experiments comparing NGSEP to state-of-the-art software using real data from three sequencing protocols and four species with different distributions of repetitive elements. NGSEP consistently shows comparative accuracy and better efficiency compared to the existing solutions. We expect that this work will contribute to the continuous improvement of quality in variant calling needed for modern applications in medicine and agriculture. AVAILABILITY AND IMPLEMENTATION: NGSEP is available as open source software at http://ngsep.sf.net. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.