Cost-effective high-throughput single-haplotype iterative mapping and sequencing for complex genomic structures
The reference sequences of structurally complex regions can be obtained only through highly accurate clone-based approaches. We and others have successfully used single-haplotype iterative mapping and sequencing (SHIMS) 1.0 to assemble structurally complex regions across the sex chromosomes of sever...
| Main Authors: | , , , , |
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| Format: | Article |
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Nature Publishing Group
2018
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| Online Access: | http://hdl.handle.net/1721.1/118374 https://orcid.org/0000-0001-9920-3411 |
| _version_ | 1811075513856294912 |
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| author | Bellott, Daniel W Cho, Ting-Jan Hughes, Jennifer F Skaletsky, Helen Page, David C |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Bellott, Daniel W Cho, Ting-Jan Hughes, Jennifer F Skaletsky, Helen Page, David C |
| author_sort | Bellott, Daniel W |
| collection | MIT |
| description | The reference sequences of structurally complex regions can be obtained only through highly accurate clone-based approaches. We and others have successfully used single-haplotype iterative mapping and sequencing (SHIMS) 1.0 to assemble structurally complex regions across the sex chromosomes of several vertebrate species and to allow for targeted improvements to the reference sequences of human autosomes. However, SHIMS 1.0 is expensive and time consuming, requiring resources that only a genome center can provide. Here we introduce SHIMS 2.0, an improved SHIMS protocol that allows even a small laboratory to generate high-quality reference sequence from complex genomic regions. Using a streamlined and parallelized library-preparation protocol, and taking advantage of inexpensive high-throughput short-read-sequencing technologies, a small laboratory with both molecular biology and bioinformatics experience can sequence and assemble 192 large-insert bacterial artificial chromosome (BAC) or fosmid clones in 1 week. In SHIMS 2.0, in contrast to other pooling strategies, each clone is sequenced with a unique barcode, thus enabling clones containing nearly identical sequences to be multiplexed in a single sequencing run and assembled separately. Relative to SHIMS 1.0, SHIMS 2.0 decreases the required cost and time by two orders of magnitude while preserving high sequencing accuracy. |
| first_indexed | 2024-09-23T10:07:29Z |
| format | Article |
| id | mit-1721.1/118374 |
| institution | Massachusetts Institute of Technology |
| last_indexed | 2024-09-23T10:07:29Z |
| publishDate | 2018 |
| publisher | Nature Publishing Group |
| record_format | dspace |
| spelling | mit-1721.1/1183742022-09-30T19:03:38Z Cost-effective high-throughput single-haplotype iterative mapping and sequencing for complex genomic structures Bellott, Daniel W Cho, Ting-Jan Hughes, Jennifer F Skaletsky, Helen Page, David C Massachusetts Institute of Technology. Department of Biology Page, David C The reference sequences of structurally complex regions can be obtained only through highly accurate clone-based approaches. We and others have successfully used single-haplotype iterative mapping and sequencing (SHIMS) 1.0 to assemble structurally complex regions across the sex chromosomes of several vertebrate species and to allow for targeted improvements to the reference sequences of human autosomes. However, SHIMS 1.0 is expensive and time consuming, requiring resources that only a genome center can provide. Here we introduce SHIMS 2.0, an improved SHIMS protocol that allows even a small laboratory to generate high-quality reference sequence from complex genomic regions. Using a streamlined and parallelized library-preparation protocol, and taking advantage of inexpensive high-throughput short-read-sequencing technologies, a small laboratory with both molecular biology and bioinformatics experience can sequence and assemble 192 large-insert bacterial artificial chromosome (BAC) or fosmid clones in 1 week. In SHIMS 2.0, in contrast to other pooling strategies, each clone is sequenced with a unique barcode, thus enabling clones containing nearly identical sequences to be multiplexed in a single sequencing run and assembled separately. Relative to SHIMS 1.0, SHIMS 2.0 decreases the required cost and time by two orders of magnitude while preserving high sequencing accuracy. 2018-10-05T15:55:43Z 2018-10-05T15:55:43Z 2018-03 2018-07-02T13:51:06Z Article http://purl.org/eprint/type/JournalArticle 1754-2189 1750-2799 http://hdl.handle.net/1721.1/118374 Bellott, Daniel W et al. “Cost-Effective High-Throughput Single-Haplotype Iterative Mapping and Sequencing for Complex Genomic Structures.” Nature Protocols 13, 4 (March 2018): 787–809 © 2018 Macmillan Publishers Limited, part of Springer Nature https://orcid.org/0000-0001-9920-3411 http://dx.doi.org/10.1038/nprot.2018.019 Nature Protocols Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf Nature Publishing Group bioRxiv |
| spellingShingle | Bellott, Daniel W Cho, Ting-Jan Hughes, Jennifer F Skaletsky, Helen Page, David C Cost-effective high-throughput single-haplotype iterative mapping and sequencing for complex genomic structures |
| title | Cost-effective high-throughput single-haplotype iterative mapping and sequencing for complex genomic structures |
| title_full | Cost-effective high-throughput single-haplotype iterative mapping and sequencing for complex genomic structures |
| title_fullStr | Cost-effective high-throughput single-haplotype iterative mapping and sequencing for complex genomic structures |
| title_full_unstemmed | Cost-effective high-throughput single-haplotype iterative mapping and sequencing for complex genomic structures |
| title_short | Cost-effective high-throughput single-haplotype iterative mapping and sequencing for complex genomic structures |
| title_sort | cost effective high throughput single haplotype iterative mapping and sequencing for complex genomic structures |
| url | http://hdl.handle.net/1721.1/118374 https://orcid.org/0000-0001-9920-3411 |
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