Starcode: sequence clustering based on all-pairs search

Zorita, Eduard; Cuscó, Pol; Filion, Guillaume J.

doi:10.1093/bioinformatics/btv053

Cited by 202 publications

(180 citation statements)

References 17 publications

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“…Here, we used improved TRIP protocols and analysis tools (Zorita et al 2015) to systematically assay the magnitude of position effects on housekeeping promoters in the Drosophila genome. We obtained a data set of ∼85,000 insertions in Kc167 cells, which, to our knowledge, is the largest of this kind to date.…”

Section: Discussionmentioning

confidence: 99%

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Clustering of Drosophila housekeeping promoters facilitates their expression

et al. 2017

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Housekeeping genes of animal genomes cluster in the same chromosomal regions. It has long been suggested that this organization contributes to their steady expression across all the tissues of the organism. Here, we show that the activity of Drosophila housekeeping gene promoters depends on the expression of their neighbors. By measuring the expression of ∼85,000 reporters integrated in Kc167 cells, we identified the best predictors of expression as chromosomal contacts with the promoters and terminators of active genes. Surprisingly, the chromatin composition at the insertion site and the contacts with enhancers were less informative. These results are substantiated by the existence of genomic "paradoxical" domains, rich in euchromatic features and enhancers, but where the reporters are expressed at low level, concomitant with a deficit of interactions with promoters and terminators. This indicates that the proper function of housekeeping genes relies not on contacts with long distance enhancers but on spatial clustering. Overall, our results suggest that spatial proximity between genes increases their expression and that the linear architecture of the Drosophila genome contributes to this effect.

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Section: Discussionmentioning

confidence: 99%

“…Barcodes were clustered using Starcode (Zorita et al 2015), allowing two errors. Contaminant reads (where the barcode belongs to another promoter library) and barcodes with less than 100 reads were removed.…”

Section: Data Sets For Bioinformatic Analysesmentioning

confidence: 99%

Clustering of Drosophila housekeeping promoters facilitates their expression

et al. 2017

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“…DNA cleavage sites were identified computationally using a cleavage scoring system described in Supplemental Figure 1. The resulting multiplex Digenome-captured sites were classified into 11 groups by edit distance (Zorita et al 2015). The computer programs used for identification of in vitro RGEN cleavage sites and classification of these Digenome-captured sites by edit distance are available at our website (www.rgenome.net/digenome).…”

Section: Whole-genome and Digenome Sequencingmentioning

confidence: 99%

Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq

et al. 2016

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We present multiplex Digenome-seq to profile genome-wide specificities of up to 11 CRISPR-Cas9 nucleases simultaneously, saving time and reducing cost. Cell-free human genomic DNA was digested using multiple sgRNAs combined with the Cas9 protein and then subjected to whole-genome sequencing. In vitro cleavage patterns, characteristic of on-and off-target sites, were computationally identified across the genome using a new DNA cleavage scoring system. We found that many falsepositive, bulge-type off-target sites were cleaved by sgRNAs transcribed from an oligonucleotide duplex but not by those transcribed from a plasmid template. Multiplex Digenome-seq captured many bona fide off-target sites, missed by other genome-wide methods, at which indels were induced at frequencies <0.1%. After analyzing 964 sites cleaved in vitro by these sgRNAs and measuring indel frequencies at hundreds of off-target sites in cells, we propose a guideline for the choice of target sites for minimizing CRISPR-Cas9 off-target effects in the human genome.

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“…This resulted in 27,822,356 total reads after merging the runs together. Barcode read counts for S0 were generated by extracting the 20 bp sequence corresponding to the barcode region from the Read 2 sequences and using Starcode 33 to collapse barcodes within a Levenshtein distance of 1 (Supp. Fig.…”

Section: Dropsynth Barcode Designmentioning

confidence: 99%

“…The resulting 294 bp amplicon was size-selected using gel-extraction, purified, pooled, and loaded onto a Hiseq 2000 single-end 50 cycle run using custom sequencing primers mi4_R1 and mi4_index, resulting in 138 million total reads. The barcodes for each sample were clustered using Starcode 33 to collapse barcodes within a Levenshtein distance of 1 (Supp. Table 1).…”

Section: Complementation Assaymentioning

confidence: 99%

Multiplexed Gene Synthesis in Emulsions for Exploring Protein Functional Landscapes

Plesa

Sidore

Lubock

et al. 2017

Preprint

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Next-generation sequencing has engendered an expanding suite of functional assays that can test sequence-function relationships at unprecedented scales in pooled formats (multiplex). Such assays are currently constrained by the short length of oligonucleotide (oligo) pools, which limit potential applications. Here we report a simple, low-cost, and scalable method called DropSynth that assembles gene libraries from oligo pools for use in multiplexed functional assays. DropSynth utilizes a library of barcoded beads to isolate and concentrate oligos needed for a gene's synthesis in a pooled format. These bead-bound oligos are then emulsified, processed, and assembled into genes within the emulsion droplets. We synthesized~1000 phylogenetically diverse orthologs of phosphopantetheine adenylyltransferase (PPAT) and tested their fitness in a multiplexed functional assay. While the majority of orthologs complement, those that do not are broadly distributed across the phylogenetic tree. Synthetic errors in our assemblies allow us to explore local landscapes around the designed orthologs revealing constrained mutations for complementing orthologs as well as gain-of-function mutations for low-fitness orthologs. This broad mutational scanning approach is complementary to deep mutational scanning and helps us understand proteins by probing evolutionarily divergent sequences that share function.peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/163550 doi: bioRxiv preprint first posted online Jul. 14, 2017;The rapid progress in DNA sequencing has provided a glimpse of the vast diversity of sequence and function that nature has explored. Unfortunately our ability to explore the functional aspects of diverse sequences remains minuscule. ) that link a function to a next generation sequencing (NGS)-based output to allow testing of thousands to millions of sequences for function in a pooled manner. Mutagenesis and deep mutational scanning combined with multiplexed functional assays are now able to systematically probe all single and double amino acid substitutions for a protein's function 3,4 . Despite the power of these approaches, the sequence space explored in such experiments is minute when compared to the evolutionary distance between even highly-homologous protein sequences. Our ability to sample a broad diversity of sequences is bottlenecked by two limitations in existing approaches for producing the libraries to be tested. First, low-cost microarray-based oligo libraries allow for large libraries of designed <200 nucleotide (nt) sequences 5 , which is far below the typical length of a protein and limits many other applications. Secondly, gene synthesis is capable of creating long-length sequences, but its costs currently make it prohibitively expensive for sampling large libraries of designed sequences [6][7][8][9] .Here we develop a new gene synthesis method we term DropSynth, a multiplexed...

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Starcode: sequence clustering based on all-pairs search

Cited by 202 publications

References 17 publications

Clustering of Drosophila housekeeping promoters facilitates their expression

Clustering of Drosophila housekeeping promoters facilitates their expression

Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq

Multiplexed Gene Synthesis in Emulsions for Exploring Protein Functional Landscapes

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