Nature Biotechnology

Here we describe single-cell corrected long-read sequencing (scCOLOR-seq), which enables error correction of barcode and unique molecular identifier oligonucleotide sequences and permits standalone cDNA nanopore sequencing of single cells. Barcodes and unique molecular identifiers are synthesized using dimeric nucleotide building blocks that allow error detection. We illustrate the use of the method for evaluating barcode assignment accuracy, differential isoform usage in myeloma cell lines, and fusion transcript detection in a sarcoma cell line.

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High-throughput mapping of a whole rhesus monkey brain at micrometer resolution

Fang Xu, Yan Shen, Lufeng Ding, Chao-Yu Yang, Heng Tan, Hao Wang, Qingyuan Zhu, Rui Xu, Fengyi Wu, Yanyang Xiao, Cheng Xu, Qianwei Li, Peng Su, Li I. Zhang, Hong-Wei Dong, Robert Desimone, Fuqiang Xu, Xintian Hu, Pak-Ming Lau & Guo-Qiang Bi

doi : 10.1038/s41587-021-00986-5

Nature Biotechnology volume 39, pages1521–1528 (2021)

Whole-brain mesoscale mapping in primates has been hindered by large brain sizes and the relatively low throughput of available microscopy methods. Here, we present an approach that combines primate-optimized tissue sectioning and clearing with ultrahigh-speed fluorescence microscopy implementing improved volumetric imaging with synchronized on-the-fly-scan and readout technique, and is capable of completing whole-brain imaging of a rhesus monkey at 1?×?1?× 2.5?µm3 voxel resolution within 100?h. We also developed a highly efficient method for long-range tracing of sparse axonal fibers in datasets numbering hundreds of terabytes. This pipeline, which we call serial sectioning and clearing, three-dimensional microscopy with semiautomated reconstruction and tracing (SMART), enables effective connectome-scale mapping of large primate brains. With SMART, we were able to construct a cortical projection map of the mediodorsal nucleus of the thalamus and identify distinct turning and routing patterns of individual axons in the cortical folds while approaching their arborization destinations.

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Cholesterol-functionalized DNA/RNA heteroduplexes cross the blood–brain barrier and knock down genes in the rodent CNS

Tetsuya Nagata, Chrissa A. Dwyer, Kie Yoshida-Tanaka, Kensuke Ihara, Masaki Ohyagi, Hidetoshi Kaburagi, Haruka Miyata, Satoe Ebihara, Kotaro Yoshioka, Takashi Ishii, Kanjiro Miyata, Kenichi Miyata, Berit Powers, Tomoko Igari, Syunsuke Yamamoto, Naoto Arimura, Hideki Hirabayashi, Toshiki Uchihara, Rintaro Iwata Hara, Takeshi Wada, C. Frank Bennett, Punit P. Seth, Frank Rigo & Takanori Yokota

doi : 10.1038/s41587-021-00972-x

Nature Biotechnology volume 39, pages1529–1536 (2021)

Achieving regulation of endogenous gene expression in the central nervous system (CNS) with antisense oligonucleotides (ASOs) administered systemically would facilitate the development of ASO-based therapies for neurological diseases. We demonstrate that DNA/RNA heteroduplex oligonucleotides (HDOs) conjugated to cholesterol or ?-tocopherol at the 5? end of the RNA strand reach the CNS after subcutaneous or intravenous administration in mice and rats. The HDOs distribute throughout the brain, spinal cord and peripheral tissues and suppress the expression of four target genes by up to 90% in the CNS, whereas single-stranded ASOs conjugated to cholesterol have limited activity. Gene knockdown was observed in major CNS cell types and was greatest in neurons and microglial cells. Side effects, such as thrombocytopenia and focal brain necrosis, were limited by using subcutaneous delivery or by dividing intravenous injections. By crossing the blood–brain barrier more effectively, cholesterol-conjugated HDOs may overcome the limited efficacy of ASOs targeting the CNS without requiring intrathecal administration.

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Enhanced detection of minimal residual disease by targeted sequencing of phased variants in circulating tumor DNA

David M. Kurtz, Joanne Soo, Lyron Co Ting Keh, Stefan Alig, Jacob J. Chabon, Brian J. Sworder, Andre Schultz, Michael C. Jin, Florian Scherer, Andrea Garofalo, Charles W. Macaulay, Emily G. Hamilton, Binbin Chen, Mari Olsen, Joseph G. Schroers-Martin, Alexander F. M. Craig, Everett J. Moding, Mohammad S. Esfahani, Chih Long Liu, Ulrich Dührsen, Andreas Hüttmann, René-Olivier Casasnovas, Jason R. Westin, Mark Roschewski, Wyndham H. Wilson, Gianluca Gaidano, Davide Rossi, Maximilian Diehn & Ash A. Alizadeh

doi : 10.1038/s41587-021-00981-w

Nature Biotechnology volume 39, pages1537–1547 (2021)

Circulating tumor-derived DNA (ctDNA) is an emerging biomarker for many cancers, but the limited sensitivity of current detection methods reduces its utility for diagnosing minimal residual disease. Here we describe phased variant enrichment and detection sequencing (PhasED-seq), a method that uses multiple somatic mutations in individual DNA fragments to improve the sensitivity of ctDNA detection. Leveraging whole-genome sequences from 2,538 tumors, we identify phased variants and their associations with mutational signatures. We show that even without molecular barcodes, the limits of detection of PhasED-seq outperform prior methods, including duplex barcoding, allowing ctDNA detection in the ppm range in participant samples. We profiled 678 specimens from 213 participants with B cell lymphomas, including serial cell-free DNA samples before and during therapy for diffuse large B cell lymphoma. In participants with undetectable ctDNA after two cycles of therapy using a next-generation sequencing-based approach termed cancer personalized profiling by deep sequencing, an additional 25% have ctDNA detectable by PhasED-seq and have worse outcomes. Finally, we demonstrate the application of PhasED-seq to solid tumors.

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Bioorthogonal information storage in L-DNA with a high-fidelity mirror-image Pfu DNA polymerase

Chuyao Fan, Qiang Deng & Ting F. Zhu

doi : 10.1038/s41587-021-00969-6

Nature Biotechnology volume 39, pages1548–1555 (2021)

Natural DNA is exquisitely evolved to store genetic information. The chirally inverted L-DNA, possessing the same informational capacity but resistant to biodegradation, may serve as a robust, bioorthogonal information repository. Here we chemically synthesize a 90-kDa high-fidelity mirror-image Pfu DNA polymerase that enables accurate assembly of a kilobase-sized mirror-image gene. We use the polymerase to encode in L-DNA an 1860 paragraph by Louis Pasteur that first proposed a mirror-image world of biology. We realize chiral steganography by embedding a chimeric D-DNA/L-DNA key molecule in a D-DNA storage library, which conveys a false or secret message depending on the chirality of reading. Furthermore, we show that a trace amount of an L-DNA barcode preserved in water from a local pond remains amplifiable and sequenceable for 1 year, whereas a D-DNA barcode under the same conditions could not be amplified after 1 day. These next-generation mirror-image molecular tools may transform the development of advanced mirror-image biology systems and pave the way for the realization of the mirror-image central dogma and exploration of their applications.

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LAMP-Seq enables sensitive, multiplexed COVID-19 diagnostics using molecular barcoding

Kerstin U. Ludwig, Ricarda M. Schmithausen, David Li, Max L. Jacobs, Ronja Hollstein, Katja Blumenstock, Jana Liebing, Miko?aj S?abicki, Amir Ben-Shmuel, Ofir Israeli, Shay Weiss, Thomas S. Ebert, Nir Paran, Wibke Rüdiger, Gero Wilbring, David Feldman, Bärbel Lippke, Nina Ishorst, Lara M. Hochfeld, Eva C. Beins, Ines H. Kaltheuner, Maximilian Schmitz, Aliona Wöhler, Manuel Döhla, Esther Sib, Marius Jentzsch, Jacob D. Borrajo, Jonathan Strecker, Julia Reinhardt, Brian Cleary, Matthias Geyer, Michael Hölzel, Rhiannon Macrae, Markus M. Nöthen, Per Hoffmann, Martin Exner, Aviv Regev, Feng Zhang & Jonathan L. Schmid-Burgk

doi : 10.1038/s41587-021-00966-9

Nature Biotechnology volume 39, pages1556–1562 (2021)

Frequent testing of large population groups combined with contact tracing and isolation measures will be crucial for containing Coronavirus Disease 2019 outbreaks. Here we present LAMP-Seq, a modified, highly scalable reverse transcription loop-mediated isothermal amplification (RT–LAMP) method. Unpurified biosamples are barcoded and amplified in a single heat step, and pooled products are analyzed en masse by sequencing. Using commercial reagents, LAMP-Seq has a limit of detection of ~2.2 molecules per µl at 95% confidence and near-perfect specificity for severe acute respiratory syndrome coronavirus 2 given its sequence readout. Clinical validation of an open-source protocol with 676 swab samples, 98 of which were deemed positive by standard RT–qPCR, demonstrated 100% sensitivity in individuals with cycle threshold values of up to 33 and a specificity of 99.7%, at a very low material cost. With a time-to-result of fewer than 24?h, low cost and little new infrastructure requirement, LAMP-Seq can be readily deployed for frequent testing as part of an integrated public health surveillance program.

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MaxDIA enables library-based and library-free data-independent acquisition proteomics

Pavel Sinitcyn, Hamid Hamzeiy, Favio Salinas Soto, Daniel Itzhak, Frank McCarthy, Christoph Wichmann, Martin Steger, Uli Ohmayer, Ute Distler, Stephanie Kaspar-Schoenefeld, Nikita Prianichnikov, ?ule Y?lmaz, Jan Daniel Rudolph, Stefan Tenzer, Yasset Perez-Riverol, Nagarjuna Nagaraj, Sean J. Humphrey & Jürgen Cox

doi : 10.1038/s41587-021-00968-7

Nature Biotechnology volume 39, pages1563–1573 (2021)

MaxDIA is a software platform for analyzing data-independent acquisition (DIA) proteomics data within the MaxQuant software environment. Using spectral libraries, MaxDIA achieves deep proteome coverage with substantially better coefficients of variation in protein quantification than other software. MaxDIA is equipped with accurate false discovery rate (FDR) estimates on both library-to-DIA match and protein levels, including when using whole-proteome predicted spectral libraries. This is the foundation of discovery DIA—hypothesis-free analysis of DIA samples without library and with reliable FDR control. MaxDIA performs three- or four-dimensional feature detection of fragment data, and scoring of matches is augmented by machine learning on the features of an identification. MaxDIA’s bootstrap DIA workflow performs multiple rounds of matching with increasing quality of recalibration and stringency of matching to the library. Combining MaxDIA with two new technologies—BoxCar acquisition and trapped ion mobility spectrometry—both lead to deep and accurate proteome quantification.

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High-content single-cell combinatorial indexing

Ryan M. Mulqueen, Dmitry Pokholok, Brendan L. O’Connell, Casey A. Thornton, Fan Zhang, Brian J. O’Roak, Jason Link, Galip Gürkan Yard?mc?, Rosalie C. Sears, Frank J. Steemers & Andrew C. Adey

doi : 10.1038/s41587-021-00962-z

Nature Biotechnology volume 39, pages1574–1580 (2021)

Single-cell combinatorial indexing (sci) with transposase-based library construction increases the throughput of single-cell genomics assays but produces sparse coverage in terms of usable reads per cell. We develop symmetrical strand sci (‘s3’), a uracil-based adapter switching approach that improves the rate of conversion of source DNA into viable sequencing library fragments following tagmentation. We apply this chemistry to assay chromatin accessibility (s3-assay for transposase-accessible chromatin, s3-ATAC) in human cortical and mouse whole-brain tissues, with mouse datasets demonstrating a six- to 13-fold improvement in usable reads per cell compared with other available methods. Application of s3 to single-cell whole-genome sequencing (s3-WGS) and to whole-genome plus chromatin conformation (s3-GCC) yields 148- and 14.8-fold improvements, respectively, in usable reads per cell compared with sci-DNA-sequencing and sci-HiC. We show that s3-WGS and s3-GCC resolve subclonal genomic alterations in patient-derived pancreatic cancer cell lines. We expect that the s3 platform will be compatible with other transposase-based techniques, including sci-MET or CUT&Tag.

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RNA demethylation increases the yield and biomass of rice and potato plants in field trials

Qiong Yu, Shun Liu, Lu Yu, Yu Xiao, Shasha Zhang, Xueping Wang, Yingying Xu, Hong Yu, Yulong Li, Junbo Yang, Jun Tang, Hong-Chao Duan, Lian-Huan Wei, Haiyan Zhang, Jiangbo Wei, Qian Tang, Chunling Wang, Wutong Zhang, Ye Wang, Peizhe Song, Qiang Lu, Wei Zhang, Shunqing Dong, Baoan Song, Chuan He & Guifang Jia

doi : 10.1038/s41587-021-00982-9

Nature Biotechnology volume 39, pages1581–1588 (2021)

RNA N6-methyladenosine (m6A) modifications are essential in plants. Here, we show that transgenic expression of the human RNA demethylase FTO in rice caused a more than threefold increase in grain yield under greenhouse conditions. In field trials, transgenic expression of FTO in rice and potato caused ~50% increases in yield and biomass. We demonstrate that the presence of FTO stimulates root meristem cell proliferation and tiller bud formation and promotes photosynthetic efficiency and drought tolerance but has no effect on mature cell size, shoot meristem cell proliferation, root diameter, plant height or ploidy. FTO mediates substantial m6A demethylation (around 7% of demethylation in poly(A) RNA and around 35% decrease of m6A in non-ribosomal nuclear RNA) in plant RNA, inducing chromatin openness and transcriptional activation. Therefore, modulation of plant RNA m6A methylation is a promising strategy to dramatically improve plant growth and crop yield.

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A pan-cancer landscape of somatic mutations in non-unique regions of the human genome

Maxime Tarabichi, Jonas Demeulemeester, Annelien Verfaillie, Adrienne M. Flanagan, Peter Van Loo & Tomasz Konopka

doi : 10.1038/s41587-021-00971-y

Nature Biotechnology volume 39, pages1589–1596 (2021)

A substantial fraction of the human genome displays high sequence similarity with at least one other genomic sequence, posing a challenge for the identification of somatic mutations from short-read sequencing data. Here we annotate genomic variants in 2,658 cancers from the Pan-Cancer Analysis of Whole Genomes (PCAWG) cohort with links to similar sites across the human genome. We train a machine learning model to use signals distributed over multiple genomic sites to call somatic events in non-unique regions and validate the data against linked-read sequencing in an independent dataset. Using this approach, we uncover previously hidden mutations in ~1,700 coding sequences and in thousands of regulatory elements, including in known cancer genes, immunoglobulins and highly mutated gene families. Mutations in non-unique regions are consistent with mutations in unique regions in terms of mutation burden and substitution profiles. The analysis provides a systematic summary of the mutation events in non-unique regions at a genome-wide scale across multiple human cancers.

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