Journal: bioRxiv

Article Title: uSort–M: Scalable isolation of user-defined sequences from diverse pooled libraries

doi: 10.64898/2026.01.12.699065

Figure Lengend Snippet: A . uSort-M converts an arbitrary pooled library of DNA variants into a “parsed” library where each variant is isolated in its own well with validated sequence information. B . uSort-M isolates single plasmid variants within a bacterial host via pooled assembly (1) and transformation (2), isolates individual bacteria via high-throughput cell sorting (FACS) (3), amplifies and barcodes DNA amplicons from cultured clones via PCR (4), and then pools amplicons for multiplexed long-read sequencing (5) that associates plate/well-specific barcodes with DNA amplicons. C . Projected costs of traditional library preparation vs . uSort-M. Left : cost as a function of library size for traditional synthesis methods (light grey shading indicates estimated minimum and maximum costs; grey line indicates mean) vs . u-Sort-M (blue line) for libraries of 300 bp fragments. Dashed black line indicates 8.3-fold savings for uSort-M vs traditional synthesis. Right : table indicating the estimated cost of individual steps required for traditional gene synthesis or uSort-M for 500- and 1,500-variant libraries, respectively, of 300 bp fragments.

Article Snippet: Sequencing costs were calculated using Plasmidsaurus Custom Sequencing pricing, which starts at $500 for 1 gigabase of sequencing and increases by $50 for each added gigabase (as of 2025; https://www.plasmidsaurus.com/custom ).

Techniques: Variant Assay, Isolation, Sequencing, Plasmid Preparation, Transformation Assay, Bacteria, High Throughput Screening Assay, FACS, Cell Culture, Clone Assay

Journal: bioRxiv

Article Title: uSort–M: Scalable isolation of user-defined sequences from diverse pooled libraries

doi: 10.64898/2026.01.12.699065

Figure Lengend Snippet: A . Schematic illustrating 328-variant human acylphosphatase 2 (hAcyP) library processing, including (1) assembly, (2) transformation, and (3) FACS sorting. Left scatter plot: selection of cell events by gating the distribution of side scatter area (SSC-A) vs . forward scatter area (FSC-A) values. Right scatter plot: selection of singlets from gating the distribution of forward scatter height (FSC-H) vs . FSC-A. B . Histogram of the number of wells as a function of measured OD 600 values. Wells with bacterial growth (green bars representing 2,057 wells) were defined as having OD 600 > 0.052 (grey vertical line). Inset pie chart indicates fraction of wells with (green, 67%) and without (grey, 33%) growth. C . Plate-well barcoding scheme for multiplexed sequencing of variant DNA in each well. Three iterations of PCR encode source plate and well location on each read. D . Number of demultiplexed MiSeq reads (sequencing depth) vs . well OD 600 ; marker color indicates plate of origin; vertical and horizontal lines are shown for OD 600 = 0.052 and sequencing depth = 100, respectively. Histogram (left) indicates number of wells with a given read depth. E . Scatter plot comparing number of reads per well from demultiplexed MiSeq data vs . Nanopore data; annotation specifies Pearson correlation coefficient. F . Read alignment plots from a representative well comparing paired-end MiSeq data and long-read Nanopore data. Sequence positions are aligned along the x-axes of each plot, with bases matching the reference are colored in light blue, mismatches in black, and empty positions in white. Top : read schematic showing aligned positions corresponding to the hAcyP2 WT reference sequence (light blue), called variant for the given well (red), and DNA appended during indexing (dark blue). Middle : Subsampled paired-end MiSeq reads ordered by orientation, with forward reads on top and reverse reads on bottom. Bottom : Subsampled Nanopore reads. G . Quantified fidelity across all reads from all wells, defined as the frequency of finding a mismatch outside of the called variant bases. Each point shows the fidelity of a single read, boxes denote the median, 25 th , and 75 th percentiles of the distribution, and error bars represent the standard deviation. H . Number of variants recovered for the 328-member library from MiSeq or Nanopore data.

Article Snippet: Sequencing costs were calculated using Plasmidsaurus Custom Sequencing pricing, which starts at $500 for 1 gigabase of sequencing and increases by $50 for each added gigabase (as of 2025; https://www.plasmidsaurus.com/custom ).

Techniques: Variant Assay, Transformation Assay, Selection, Sequencing, Marker, Standard Deviation

Journal: bioRxiv

Article Title: uSort–M: Scalable isolation of user-defined sequences from diverse pooled libraries

doi: 10.64898/2026.01.12.699065

Figure Lengend Snippet: A . Simulation pipeline illustrating key parameters from each step in the uSort-M workflow and how they are defined. B . Simulated coverage as a function of fold-sampling of a 328-member library, where the number of wells sorted is equal to the product of the library size and fold-sampling. Red points represent bootstrap downsampling from the collected hAcyP2 library sequencing data for each fold-sampling value; blue shaded area shows 99% confidence interval from 100 independent simulations using known parameters of the experiment (skew = 2, off-target variation = 0.35, transformation scale = 5,250/328, sorting efficiency = 0.67, PCR failure rate = 0.025). Grey shaded area shows 99% confidence interval from 100 independent numerical simulations assuming a ‘perfect’ input library (skew = 1, off-target variation = 0, transformation scale = 100, same sorting and PCR parameters); dashed black line indicates the analytical solution to sampling from this population. Inset shows simulated coverage distribution at 3,072 sorted wells in blue; red line indicates the observed coverage (0.963, 316 variants). C . Coverage vs . fold-sampling plots for a 1000-member library with varying skew, off-target variation, and transformation scale, holding non-varied parameters at ‘realistic’ values of 1, 0, and 50, respectively. The x-axis is discontinuous between 10x sampling and >380x sampling to illustrate where the curves plateau. D . Sampling vs . coverage ( upper ) or vs . cost ( lower ) for either exhaustive sampling (blue curve) or sampling with a ‘targeted resynthesis’ step (orange curve) of a 1,000-member library. In ‘targeted resynthesis’, a set number of wells are sorted and sequenced to a lower coverage value, and then the remaining unsampled members of the library are reordered as a new, smaller library.

Article Snippet: Sequencing costs were calculated using Plasmidsaurus Custom Sequencing pricing, which starts at $500 for 1 gigabase of sequencing and increases by $50 for each added gigabase (as of 2025; https://www.plasmidsaurus.com/custom ).

Techniques: Sampling, Sequencing, Transformation Assay

Journal: Communications Biology

Article Title: A realistic FastQ-based framework FastQDesign for ScRNA-seq study design issues

doi: 10.1038/s42003-025-07938-8

Figure Lengend Snippet: a–c The UMAPs of the FastQ sample with 10% read depth of all cells, the UMI sample with 10% read depth of all cells, 10% cell and total read depth, were colored with the consistency of cell clustering match with the reference. d–f The pairwise minimal p-value 's of each gene among the clusters from the samples and the reference, x is \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$-\log 10(p\_val\_adj)$$\end{document} − log 10 ( p _ v a l _ a d j ) in the reference, y is the same value in the sample, only the significant DE genes in either reference or samples are shown. g–i The pairwise minimal p-value 's of each gene between the condition within the cluster from the samples and the reference, only the significant DE genes in either reference or samples are shown. j, k The impact of varying read depth, and cell numbers on ARI and Jaccard indexes, 10 simulations were conducted at each setting.

Article Snippet: The overall cost \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$g({N}^{{\prime} },{R}^{{\prime} })$$\end{document} g ( N ′ , R ′ ) for a 10X Genomics experiment is composed of library preparation cost( C p r e p ), and the sequencing cost( C s e q ) for a flow cell with the read capacity of a . Library preparation: Multiple samples can be prepared in the same library by using feature barcode technology (CellPlex kit).

Techniques:

Journal: bioRxiv

Article Title: Labor- and cost-effective long-read amplicon sequencing using a plasmid analysis service: Application to transposon-inserted alleles in Japanese morning glory

doi: 10.1101/2024.09.19.613814

Figure Lengend Snippet: Flowchart for the preparation of templates from PCR amplicons for whole plasmid DNA sequencing services using Nanopore sequencers. T4PNK; T4 Polynucleotide Kinase.

Article Snippet: However, low-cost full-length plasmid DNA sequencing services using Nanopore technology have recently been launched by several companies (e.g., Eurofins Genomics (USA), AZENTA Life Sciences (USA), and Plasmidsaurus (USA)) and are currently available in Japan.

Techniques: Plasmid Preparation, DNA Sequencing

Journal: bioRxiv

Article Title: Labor- and cost-effective long-read amplicon sequencing using a plasmid analysis service: Application to transposon-inserted alleles in Japanese morning glory

doi: 10.1101/2024.09.19.613814

Figure Lengend Snippet: Flowchart for the preparation of templates from PCR amplicons for whole plasmid DNA sequencing services using Nanopore sequencers. T4PNK; T4 Polynucleotide Kinase.

Article Snippet: However, low-cost full-length plasmid DNA sequencing services using Nanopore technology have recently been launched by several companies (e.g., Eurofins Genomics (USA), AZENTA Life Sciences (USA), and Plasmidsaurus (USA)) and are currently available in Japan.

Techniques: Plasmid Preparation, DNA Sequencing