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Using high-throughput barcode sequencing to efficiently map connectomes.

ABSTRACT: The function of a neural circuit is determined by the details of its synaptic connections. At present, the only available method for determining a neural wiring diagram with single synapse precision-a 'connectome'-is based on imaging methods that are slow, labor-intensive and expensive. Here, we present SYNseq, a method for converting the connectome into a form that can exploit the speed and low cost of modern high-throughput DNA sequencing. In SYNseq, each neuron is labeled with a unique random nucleotide sequence-an RNA 'barcode'-which is targeted to the synapse using engineered proteins. Barcodes in pre- and postsynaptic neurons are then associated through protein-protein crosslinking across the synapse, extracted from the tissue, and joined into a form suitable for sequencing. Although our failure to develop an efficient barcode joining scheme precludes the widespread application of this approach, we expect that with further development SYNseq will enable tracing of complex circuits at high speed and low cost.

SUBMITTER: Peikon ID

PROVIDER: S-EPMC5499584 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Using high-throughput barcode sequencing to efficiently map connectomes.

Peikon Ian D ID Kebschull Justus M JM Vagin Vasily V VV Ravens Diana I DI Sun Yu-Chi YC Brouzes Eric E Corrêa Ivan R IR Bressan Dario D Zador Anthony M AM

Nucleic acids research 20170701 12

The function of a neural circuit is determined by the details of its synaptic connections. At present, the only available method for determining a neural wiring diagram with single synapse precision-a 'connectome'-is based on imaging methods that are slow, labor-intensive and expensive. Here, we present SYNseq, a method for converting the connectome into a form that can exploit the speed and low cost of modern high-throughput DNA sequencing. In SYNseq, each neuron is labeled with a unique random ...[more]

PMID: 28449067

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Project description:BackgroundCurrent high-throughput sequencing platforms provide capacity to sequence multiple samples in parallel. Different samples are labeled by attaching a short sample specific nucleotide sequence, barcode, to each DNA molecule prior pooling them into a mix containing a number of libraries to be sequenced simultaneously. After sequencing, the samples are binned by identifying the barcode sequence within each sequence read. In order to tolerate sequencing errors, barcodes should be sufficiently apart from each other in sequence space. An additional constraint due to both nucleotide usage and basecalling accuracy is that the proportion of different nucleotides should be in balance in each barcode position. The number of samples to be mixed in each sequencing run may vary and this introduces a problem how to select the best subset of available barcodes at sequencing core facility for each sequencing run. There are plenty of tools available for de novo barcode design, but they are not suitable for subset selection.ResultsWe have developed a tool which can be used for three different tasks: 1) selecting an optimal barcode set from a larger set of candidates, 2) checking the compatibility of user-defined set of barcodes, e.g. whether two or more libraries with existing barcodes can be combined in a single sequencing pool, and 3) augmenting an existing set of barcodes. In our approach the selection process is formulated as a minimization problem. We define the cost function and a set of constraints and use integer programming to solve the resulting combinatorial problem. Based on the desired number of barcodes to be selected and the set of candidate sequences given by user, the necessary constraints are automatically generated and the optimal solution can be found. The method is implemented in C programming language and web interface is available at http://ekhidna2.biocenter.helsinki.fi/barcosel .ConclusionsIncreasing capacity of sequencing platforms raises the challenge of mixing barcodes. Our method allows the user to select a given number of barcodes among the larger existing barcode set so that both sequencing errors are tolerated and the nucleotide balance is optimized. The tool is easy to access via web browser.

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Using high-throughput barcode sequencing to efficiently map connectomes.

Publications

Using high-throughput barcode sequencing to efficiently map connectomes.

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