scholarly journals Efficient in situ barcode sequencing using padlock probe-based BaristaSeq

2017 ◽  
Vol 46 (4) ◽  
pp. e22-e22 ◽  
Author(s):  
Xiaoyin Chen ◽  
Yu-Chi Sun ◽  
George M Church ◽  
Je Hyuk Lee ◽  
Anthony M Zador
Keyword(s):  
Padlock Probe ◽  
Barcode Sequencing

scholarly journals Efficient in situ barcode sequencing using padlock probe-based BaristaSeq

2017 ◽  
Author(s):  
Xiaoyin Chen ◽  
Yu-Chi Sun ◽  
George M Church ◽  
Je Hyuk Lee ◽  
Anthony M Zador
Keyword(s):  
Spatial Resolution ◽  
High Throughput ◽  
Illumina Sequencing ◽  
Lineage Tracing ◽  
List Type ◽  
Padlock Probe ◽  
Gap Filling ◽  
Sequencing Chemistry ◽  
Barcode Sequencing

AbstractCellular DNA/RNA tags (barcodes) allow for multiplexed cell lineage tracing and neuronal projection mapping with cellular resolution. Conventional approaches to reading out cellular barcodes trade off spatial resolution with throughput. Bulk sequencing achieves high throughput but sacrifices spatial resolution, whereas manual cell picking has low throughput. In situ sequencing could potentially achieve both high spatial resolution and high throughput, but current in situ sequencing techniques are inefficient at reading out cellular barcodes. Here we describe BaristaSeq, an optimization of a targeted, padlock probe-based technique for in situ barcode sequencing compatible with Illumina sequencing chemistry. BaristaSeq results in a five-fold increase in amplification efficiency, with a sequencing accuracy of at least 97%. BaristaSeq could be used for barcode-assisted lineage tracing, and to map long-range neuronal projections.Key PointsIn situ sequencing by gap-filling padlock probes is limited by the strand displacement of DNA polymerasesIllumina sequencing chemistry offers superior signal-to-noise ratio in situ compared to sequencing by ligationBaristaSeq as an accurate method for barcode sequencing in situ with improved gap-filling efficiency

scholarly journals In Situ Detection and Quantification of AR-V7, AR-FL, PSA, and KRAS Point Mutations in Circulating Tumor Cells

2018 ◽  
Vol 64 (3) ◽  
pp. 536-546 ◽  
Author(s):  
Amin El-Heliebi ◽  
Claudia Hille ◽  
Navya Laxman ◽  
Jessica Svedlund ◽  
Christoph Haudum ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Specific Antigen ◽  
Padlock Probe ◽  
Castration Resistant Prostate Cancer ◽  
Kras Mutations ◽  
Padlock Probes ◽  
Kras Status

Abstract BACKGROUND Liquid biopsies can be used in castration-resistant prostate cancer (CRPC) to detect androgen receptor splice variant 7 (AR-V7), a splicing product of the androgen receptor. Patients with AR-V7-positive circulating tumor cells (CTCs) have greater benefit of taxane chemotherapy compared with novel hormonal therapies, indicating a treatment-selection biomarker. Likewise, in those with pancreatic cancer (PaCa), KRAS mutations act as prognostic biomarkers. Thus, there is an urgent need for technology investigating the expression and mutation status of CTCs. Here, we report an approach that adds AR-V7 or KRAS status to CTC enumeration, compatible with multiple CTC-isolation platforms. METHODS We studied 3 independent CTC-isolation devices (CellCollector, Parsortix, CellSearch) for the evaluation of AR-V7 or KRAS status of CTCs with in situ padlock probe technology. Padlock probes allow highly specific detection and visualization of transcripts on a cellular level. We applied padlock probes for detecting AR-V7, androgen receptor full length (AR-FL), and prostate-specific antigen (PSA) in CRPC and KRAS wild-type (wt) and mutant (mut) transcripts in PaCa in CTCs from 46 patients. RESULTS In situ analysis showed that 71% (22 of 31) of CRPC patients had detectable AR-V7 expression ranging from low to high expression [1–76 rolling circle products (RCPs)/CTC]. In PaCa patients, 40% (6 of 15) had KRAS mut expressing CTCs with 1 to 8 RCPs/CTC. In situ padlock probe analysis revealed CTCs with no detectable cytokeratin expression but positivity for AR-V7 or KRAS mut transcripts. CONCLUSIONS Padlock probe technology enables quantification of AR-V7, AR-FL, PSA, and KRAS mut/wt transcripts in CTCs. The technology is easily applicable in routine laboratories and compatible with multiple CTC-isolation devices.

scholarly journals Fluorescent in situ sequencing of DNA barcoded antibodies

2020 ◽  
Author(s):  
Richie E. Kohman ◽  
George M. Church
Keyword(s):  
Signal Amplification ◽  
New Technology ◽  
Biological Tissues ◽  
Antibody Staining ◽  
Amplification Method ◽  
And Function ◽  
Conventional Antibody ◽  
Barcode Sequencing ◽  
Insight Into

AbstractBiological tissues contain thousands of different proteins yet conventional antibody staining can only assay a few at a time because of the limited number of spectrally distinct fluorescent labels. The capacity to map the location of hundreds or thousands of proteins within a single sample would allow for an unprecedented investigation of the spatial proteome, and give insight into the development and function of diseased and healthy tissues. In order to achieve this goal, we propose a new technology that leverages established methodologies for in situ imaging of nucleic acids to achieve near limitless multiplexing. The exponential scaling power of DNA technologies ties multiplexing to the number of DNA nucleotides sequenced rather than the number of spectrally distinct labels. Here we demonstrate that barcode sequencing can be applied to in situ proteomics by sequencing DNA conjugated antibodies bound to biological samples. In addition, we show a signal amplification method which is compatible with barcoded antibodies.

Targeted Expansion Sequencing Protocols v2

2021 ◽  
Author(s):  
Anubhav Sinha ◽  
Yi Cui ◽  
Shahar Alon ◽  
Fei Chen ◽  
Asmamaw T. Wassie ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Human Tumor ◽  
Metastatic Breast ◽  
Pilot Project ◽  
Probe Design ◽  
Tissue Preparation ◽  
Padlock Probe ◽  
Library Preparation ◽  
Key Steps

This protocol collection accompanies accompanies Expansion Sequencing (ExSeq), covering the four key steps of a targeted Expansion Sequencing (targeted ExSeq) experiment: (1) Padlock probe design; (2) tissue preparation and expansion; (3) library preparation; and (4) in situ sequencing with the Illumina chemistry. For further details, consult the relevant protocols within the collection. These protocols were used to profile human metastatic breast cancer biopsies as a part of the Human Tumor Atlas Pilot Project (HTAPP).

scholarly journals Visualization of tumor heterogeneity by in situ padlock probe technology in colorectal cancer

2017 ◽  
Vol 148 (2) ◽  
pp. 105-115 ◽  
Author(s):  
Amin El-Heliebi ◽  
Karl Kashofer ◽  
Julia Fuchs ◽  
Stephan W. Jahn ◽  
Christian Viertler ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Tumor Heterogeneity ◽  
Padlock Probe

Targeted Expansion Sequencing Protocols v3

2021 ◽  
Author(s):  
Anubhav Sinha ◽  
Yi Cui ◽  
Shahar Alon ◽  
Fei Chen ◽  
Asmamaw T. Wassie ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Human Tumor ◽  
Metastatic Breast ◽  
Pilot Project ◽  
Probe Design ◽  
Tissue Preparation ◽  
Padlock Probe ◽  
Library Preparation ◽  
Key Steps

This protocol collection accompanies accompanies Expansion Sequencing (ExSeq), covering the four key steps of a targeted Expansion Sequencing (targeted ExSeq) experiment: (1) Padlock probe design; (2) tissue preparation and expansion; (3) library preparation; and (4) in situ sequencing with the Illumina chemistry. For further details, consult the relevant protocols within the collection. These protocols were used to profile human metastatic breast cancer biopsies as a part of the Human Tumor Atlas Pilot Project (HTAPP).

Rendez vous with Saturn's rings

1984 ◽  
Vol 75 ◽  
pp. 743-759 ◽  
Author(s):  
Kerry T. Nock
Keyword(s):  
Solar Energy ◽  
Electric Propulsion ◽  
Power Source ◽  
Propulsion System ◽  
Low Thrust ◽  
Ring Plane ◽  
The Earth ◽  
Total Impulse ◽  
Saturn's Rings

ABSTRACTA mission to rendezvous with the rings of Saturn is studied with regard to science rationale and instrumentation and engineering feasibility and design. Future detailedin situexploration of the rings of Saturn will require spacecraft systems with enormous propulsive capability. NASA is currently studying the critical technologies for just such a system, called Nuclear Electric Propulsion (NEP). Electric propulsion is the only technology which can effectively provide the required total impulse for this demanding mission. Furthermore, the power source must be nuclear because the solar energy reaching Saturn is only 1% of that at the Earth. An important aspect of this mission is the ability of the low thrust propulsion system to continuously boost the spacecraft above the ring plane as it spirals in toward Saturn, thus enabling scientific measurements of ring particles from only a few kilometers.

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