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Nature Biotechnology

Shu-Qi Zhang, Ke-Yue Ma, Alexandra A Schonnesen, Mingliang Zhang, Chenfeng He, Eric Sun, Chad M Williams, Weiping Jia, Ning Jiang
We present tetramer-associated T-cell receptor sequencing (TetTCR-seq) to link T cell receptor (TCR) sequences to their cognate antigens in single cells at high throughput. Binding is determined using a library of DNA-barcoded antigen tetramers that is rapidly generated by in vitro transcription and translation. We applied TetTCR-seq to identify patterns in TCR cross-reactivity with cancer neoantigens and to rapidly isolate neoantigen-specific TCRs with no cross-reactivity to the wild-type antigen.
November 12, 2018: Nature Biotechnology
Sara H Rouhanifard, Ian A Mellis, Margaret Dunagin, Sareh Bayatpour, Connie L Jiang, Ian Dardani, Orsolya Symmons, Benjamin Emert, Eduardo Torre, Allison Cote, Alessandra Sullivan, John A Stamatoyannopoulos, Arjun Raj
Methods for detecting single nucleic acids in cell and tissues, such as fluorescence in situ hybridization (FISH), are limited by relatively low signal intensity and nonspecific probe binding. Here we present click-amplifying FISH (clampFISH), a method for fluorescence detection of nucleic acids that achieves high specificity and high-gain (>400-fold) signal amplification. ClampFISH probes form a 'C' configuration upon hybridization to the sequence of interest in a double helical manner. The ends of the probes are ligated together using bio-orthogonal click chemistry, effectively locking the probes around the target...
November 12, 2018: Nature Biotechnology
Ying Liu, Zhongzheng Cao, Yinan Wang, Yu Guo, Ping Xu, Pengfei Yuan, Zhiheng Liu, Yuan He, Wensheng Wei
The functions of many long noncoding RNAs (lncRNAs) in the human genome remain unknown owing to the lack of scalable loss-of-function screening tools. We previously used pairs of CRISPR-Cas9 (refs. 1, 2, 3) single guide RNAs (sgRNAs) for small-scale functional screening of lncRNAs. Here we demonstrate genome-wide screening of lncRNA function using sgRNAs to target splice sites and achieve exon skipping or intron retention. Splice-site targeting outperformed a conventional CRISPR library in a negative selection screen targeting 79 ribosomal genes...
November 5, 2018: Nature Biotechnology
Qian Zhu, Sheel Shah, Ruben Dries, Long Cai, Guo-Cheng Yuan
How intrinsic gene-regulatory networks interact with a cell's spatial environment to define its identity remains poorly understood. We developed an approach to distinguish between intrinsic and extrinsic effects on global gene expression by integrating analysis of sequencing-based and imaging-based single-cell transcriptomic profiles, using cross-platform cell type mapping combined with a hidden Markov random field model. We applied this approach to dissect the cell-type- and spatial-domain-associated heterogeneity in the mouse visual cortex region...
October 29, 2018: Nature Biotechnology
Victoria Yao, Rachel Kaletsky, William Keyes, Danielle E Mor, Aaron K Wong, Salman Sohrabi, Coleen T Murphy, Olga G Troyanskaya
Effective discovery of causal disease genes must overcome the statistical challenges of quantitative genetics studies and the practical limitations of human biology experiments. Here we developed diseaseQUEST, an integrative approach that combines data from human genome-wide disease studies with in silico network models of tissue- and cell-type-specific function in model organisms to prioritize candidates within functionally conserved processes and pathways. We used diseaseQUEST to predict candidate genes for 25 different diseases and traits, including cancer, longevity, and neurodegenerative diseases...
October 22, 2018: Nature Biotechnology
Hila Wolf-Levy, Aaron Javitt, Avital Eisenberg-Lerner, Assaf Kacen, Adi Ulman, Daoud Sheban, Bareket Dassa, Vered Fishbain-Yoskovitz, Carmelo Carmona-Rivera, Matthias P Kramer, Neta Nudel, Ifat Regev, Liron Zahavi, Dalia Elinger, Mariana J Kaplan, David Morgenstern, Yishai Levin, Yifat Merbl
Cellular function is critically regulated through degradation of substrates by the proteasome. To enable direct analysis of naturally cleaved proteasomal peptides under physiological conditions, we developed mass spectrometry analysis of proteolytic peptides (MAPP), a method for proteasomal footprinting that allows for capture, isolation and analysis of proteasome-cleaved peptides. Application of MAPP to cancer cell lines as well as primary immune cells revealed dynamic modulation of the cellular degradome in response to various stimuli, such as proinflammatory signals...
October 22, 2018: Nature Biotechnology
Sergey Koren, Arang Rhie, Brian P Walenz, Alexander T Dilthey, Derek M Bickhart, Sarah B Kingan, Stefan Hiendleder, John L Williams, Timothy P L Smith, Adam M Phillippy
Complex allelic variation hampers the assembly of haplotype-resolved sequences from diploid genomes. We developed trio binning, an approach that simplifies haplotype assembly by resolving allelic variation before assembly. In contrast with prior approaches, the effectiveness of our method improved with increasing heterozygosity. Trio binning uses short reads from two parental genomes to first partition long reads from an offspring into haplotype-specific sets. Each haplotype is then assembled independently, resulting in a complete diploid reconstruction...
October 22, 2018: Nature Biotechnology
Jagannath Swaminathan, Alexander A Boulgakov, Erik T Hernandez, Angela M Bardo, James L Bachman, Joseph Marotta, Amber M Johnson, Eric V Anslyn, Edward M Marcotte
The identification and quantification of proteins lags behind DNA-sequencing methods in scale, sensitivity, and dynamic range. Here, we show that sparse amino acid-sequence information can be obtained for individual protein molecules for thousands to millions of molecules in parallel. We demonstrate selective fluorescence labeling of cysteine and lysine residues in peptide samples, immobilization of labeled peptides on a glass surface, and imaging by total internal reflection microscopy to monitor decreases in each molecule's fluorescence after consecutive rounds of Edman degradation...
October 22, 2018: Nature Biotechnology
Ishaan Gupta, Paul G Collier, Bettina Haase, Ahmed Mahfouz, Anoushka Joglekar, Taylor Floyd, Frank Koopmans, Ben Barres, August B Smit, Steven A Sloan, Wenjie Luo, Olivier Fedrigo, M Elizabeth Ross, Hagen U Tilgner
Full-length RNA sequencing (RNA-Seq) has been applied to bulk tissue, cell lines and sorted cells to characterize transcriptomes, but applying this technology to single cells has proven to be difficult, with less than ten single-cell transcriptomes having been analyzed thus far. Although single splicing events have been described for ≤200 single cells with statistical confidence, full-length mRNA analyses for hundreds of cells have not been reported. Single-cell short-read 3' sequencing enables the identification of cellular subtypes, but full-length mRNA isoforms for these cell types cannot be profiled...
October 15, 2018: Nature Biotechnology
Alex Bishara, Eli L Moss, Mikhail Kolmogorov, Alma E Parada, Ziming Weng, Arend Sidow, Anne E Dekas, Serafim Batzoglou, Ami S Bhatt
Although shotgun metagenomic sequencing of microbiome samples enables partial reconstruction of strain-level community structure, obtaining high-quality microbial genome drafts without isolation and culture remains difficult. Here, we present an application of read clouds, short-read sequences tagged with long-range information, to microbiome samples. We present Athena, a de novo assembler that uses read clouds to improve metagenomic assemblies. We applied this approach to sequence stool samples from two healthy individuals and compared it with existing short-read and synthetic long-read metagenomic sequencing techniques...
October 15, 2018: Nature Biotechnology
Min-Jung Kwak, Hyun Gi Kong, Kihyuck Choi, Soon-Kyeong Kwon, Ju Yeon Song, Jidam Lee, Pyeong An Lee, Soo Yeon Choi, Minseok Seo, Hyoung Ju Lee, Eun Joo Jung, Hyein Park, Nazish Roy, Heebal Kim, Myeong Min Lee, Edward M Rubin, Seon-Woo Lee, Jihyun F Kim
Tomato variety Hawaii 7996 is resistant to the soil-borne pathogen Ralstonia solanacearum, whereas the Moneymaker variety is susceptible to the pathogen. To evaluate whether plant-associated microorganisms have a role in disease resistance, we analyzed the rhizosphere microbiomes of both varieties in a mesocosm experiment. Microbiome structures differed between the two cultivars. Transplantation of rhizosphere microbiota from resistant plants suppressed disease symptoms in susceptible plants. Comparative analyses of rhizosphere metagenomes from resistant and susceptible plants enabled the identification and assembly of a flavobacterial genome that was far more abundant in the resistant plant rhizosphere microbiome than in that of the susceptible plant...
October 8, 2018: Nature Biotechnology
Emily K Schutsky, Jamie E DeNizio, Peng Hu, Monica Yun Liu, Christopher S Nabel, Emily B Fabyanic, Young Hwang, Frederic D Bushman, Hao Wu, Rahul M Kohli
Here we present APOBEC-coupled epigenetic sequencing (ACE-seq), a bisulfite-free method for localizing 5-hydroxymethylcytosine (5hmC) at single-base resolution with low DNA input. The method builds on the observation that AID/APOBEC family DNA deaminase enzymes can potently discriminate between cytosine modification states and exploits the non-destructive nature of enzymatic, rather than chemical, deamination. ACE-seq yielded high-confidence 5hmC profiles with at least 1,000-fold less DNA input than conventional methods...
October 8, 2018: Nature Biotechnology
Hao Chi, Chao Liu, Hao Yang, Wen-Feng Zeng, Long Wu, Wen-Jing Zhou, Rui-Min Wang, Xiu-Nan Niu, Yue-He Ding, Yao Zhang, Zhao-Wei Wang, Zhen-Lin Chen, Rui-Xiang Sun, Tao Liu, Guang-Ming Tan, Meng-Qiu Dong, Ping Xu, Pei-Heng Zhang, Si-Min He
We present a sequence-tag-based search engine, Open-pFind, to identify peptides in an ultra-large search space that includes coeluting peptides, unexpected modifications and digestions. Our method detects peptides with higher precision and speed than seven other search engines. Open-pFind identified 70-85% of the tandem mass spectra in four large-scale datasets and 14,064 proteins, each supported by at least two protein-unique peptides, in a human proteome dataset.
October 8, 2018: Nature Biotechnology
Yuan Zong, Qianna Song, Chao Li, Shuai Jin, Dingbo Zhang, Yanpeng Wang, Jin-Long Qiu, Caixia Gao
Base editors (BEs) have been used to create C-to-T substitutions in various organisms. However, editing with rat APOBEC1-based BE3 is limited to a 5-nt sequence editing window and is inefficient in GC contexts. Here, we show that a base editor fusion protein composed of Cas9 nickase and human APOBEC3A (A3A-PBE) converts cytidine to thymidine efficiently in wheat, rice and potato with a 17-nucleotide editing window at all examined sites, independent of sequence context.
October 1, 2018: Nature Biotechnology
Agustin Zsögön, Tomáš Čermák, Emmanuel Rezende Naves, Marcela Morato Notini, Kai H Edel, Stefan Weinl, Luciano Freschi, Daniel F Voytas, Jörg Kudla, Lázaro Eustáquio Pereira Peres
Breeding of crops over millennia for yield and productivity has led to reduced genetic diversity. As a result, beneficial traits of wild species, such as disease resistance and stress tolerance, have been lost. We devised a CRISPR-Cas9 genome engineering strategy to combine agronomically desirable traits with useful traits present in wild lines. We report that editing of six loci that are important for yield and productivity in present-day tomato crop lines enabled de novo domestication of wild Solanum pimpinellifolium...
October 1, 2018: Nature Biotechnology
Laura E Crowell, Amos E Lu, Kerry R Love, Alan Stockdale, Steven M Timmick, Di Wu, Yu Annie Wang, William Doherty, Alexandra Bonnyman, Nicholas Vecchiarello, Chaz Goodwine, Lisa Bradbury, Joseph R Brady, John J Clark, Noelle A Colant, Aleksandar Cvetkovic, Neil C Dalvie, Diana Liu, Yanjun Liu, Craig A Mascarenhas, Catherine B Matthews, Nicholas J Mozdzierz, Kartik A Shah, Shiaw-Lin Wu, William S Hancock, Richard D Braatz, Steven M Cramer, J Christopher Love
Conventional manufacturing of protein biopharmaceuticals in centralized, large-scale, single-product facilities is not well-suited to the agile production of drugs for small patient populations or individuals. Previous solutions for small-scale manufacturing are limited in both process reproducibility and product quality, owing to their complicated means of protein expression and purification. We describe an automated, benchtop, multiproduct manufacturing system, called Integrated Scalable Cyto-Technology (InSCyT), for the end-to-end production of hundreds to thousands of doses of clinical-quality protein biologics in about 3 d...
October 1, 2018: Nature Biotechnology
Tingdong Li, Xinping Yang, Yuan Yu, Xiaomin Si, Xiawan Zhai, Huawei Zhang, Wenxia Dong, Caixia Gao, Cao Xu
Crop improvement by inbreeding often results in fitness penalties and loss of genetic diversity. We introduced desirable traits into four stress-tolerant wild-tomato accessions by using multiplex CRISPR-Cas9 editing of coding sequences, cis-regulatory regions or upstream open reading frames of genes associated with morphology, flower and fruit production, and ascorbic acid synthesis. Cas9-free progeny of edited plants had domesticated phenotypes yet retained parental disease resistance and salt tolerance.
October 1, 2018: Nature Biotechnology
Nicolas Fiorini, Robert Leaman, David J Lipman, Zhiyong Lu
PubMed is a widely used search engine for biomedical literature. It is developed and maintained by the US National Library of Medicine/National Center for Biotechnology Information and is visited daily by millions of users around the world. For decades, PubMed has used advanced artificial intelligence technologies that extract patterns of collective user activity, such as machine learning and natural language processing, to inform the algorithmic changes that ultimately improve a user's search experience. Although these efforts have led to objective improvements in search quality, the technical underpinnings remain largely invisible and go largely unnoticed by most users...
October 1, 2018: Nature Biotechnology
Kyros Kyrou, Andrew M Hammond, Roberto Galizi, Nace Kranjc, Austin Burt, Andrea K Beaghton, Tony Nolan, Andrea Crisanti
In the human malaria vector Anopheles gambiae, the gene doublesex (Agdsx) encodes two alternatively spliced transcripts, dsx-female (AgdsxF) and dsx-male (AgdsxM), that control differentiation of the two sexes. The female transcript, unlike the male, contains an exon (exon 5) whose sequence is highly conserved in all Anopheles mosquitoes so far analyzed. We found that CRISPR-Cas9-targeted disruption of the intron 4-exon 5 boundary aimed at blocking the formation of functional AgdsxF did not affect male development or fertility, whereas females homozygous for the disrupted allele showed an intersex phenotype and complete sterility...
December 2018: Nature Biotechnology
Alicia C Smart, Claire A Margolis, Harold Pimentel, Meng Xiao He, Diana Miao, Dennis Adeegbe, Tim Fugmann, Kwok-Kin Wong, Eliezer M Van Allen
We present an in silico approach to identifying neoepitopes derived from intron retention events in tumor transcriptomes. Using mass spectrometry immunopeptidome analysis, we show that retained intron neoepitopes are processed and presented on MHC I on the surface of cancer cell lines. RNA-derived neoepitopes should be considered for prospective personalized cancer vaccine development.
December 2018: Nature Biotechnology
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