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Old 10-24-2010, 12:11 PM   #1
k-gun12
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Default Chromosomal isolation & sequencing

Wondering if anyone has seen, used or developed a good protocol for chromosomal isolation and sequencing via NGS. I've seen flow cytometry protocols around (Ibrahim et al. 2004). I've got access to FACS sorters but I'm unsure of their hardware right now. Anyone have anything more recent?

Thanks!
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Old 10-24-2010, 01:32 PM   #2
k-gun12
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I should also mention I'm also starting from a live insect larva.. not a cell line.
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Old 10-24-2010, 07:51 PM   #3
krobison
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The one paper I'm aware of

Deep short-read sequencing of chromosome 17 from the mouse strains A/J and CAST/Ei identifies significant germline variation and candidate genes that regulate liver triglyceride levels
Ian Sudbery1¤, Jim Stalker1¤, Jared T Simpson1, Thomas Keane1, Alistair G Rust1, Matthew E Hurles1, Klaudia Walter1, Dee Lynch2, Lydia Teboul2, Steve D Brown2, Heng Li1, Zemin Ning1, Joseph H Nadeau3, Colleen M Croniger3, Richard Durbin1 and David J Adams1*
* Corresponding author: David J Adams da1@sanger.ac.uk
¤ Equal contributors
Author Affiliations
1 The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, CB10 1HH, UK
2 Mammalian Genetics Unit, MRC-Harwell, Harwell Science and Innovation Campus, Oxfordshire, OX11 ORD, UK
3 Department of Genetics, Case Western Reserve University, Adelbert Rd, Cleveland, OH 44106-4955. USA

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Genome Biology 2009, 10:R112 doi:10.1186/gb-2009-10-10-r112

The electronic version of this article is the complete one and can be found online at: http://genomebiology.com/2009/10/10/R112

Received: 5 July 2009
Revisions received: 26 August 2009
Accepted: 13 October 2009
Published: 13 October 2009

© 2009 Sudbery et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited
Abstract
Genome sequences are essential tools for comparative and mutational analyses. Here we present the short read sequence of mouse chromosome 17 from the Mus musculus domesticus derived strain A/J, and the Mus musculus castaneus derived strain CAST/Ei. We describe approaches for the accurate identification of nucleotide and structural variation in the genomes of vertebrate experimental organisms, and show how these techniques can be applied to help prioritize candidate genes within quantitative trait loci.

Background
Mouse genetics has its origins with mouse fanciers who bred and maintained individuals lines of mice because of their unusual coat color or behavior [1,2] . The derivation of the classical laboratory mouse strains, however, only commenced early last century, driven by a desire to model human disease mechanisms [3] . It is now clear that these classical mouse strains are derived from a common pool of founders because haplotypes are shared, but have been shuffled, between strains prior to being fixed by inbreeding to select for desirable trait characteristics [4-7] . In contrast, wild-derived strains are highly divergent from these classical strains of mice, having been founded by inbreeding of wild-derived isolates [8] .

In addition to the sequence of the reference strain, C57BL/6J, there are two large resources for genomic sequence of inbred mouse strains. Four laboratory strains were included by Celera in a whole genomic shotgun sequence of the mouse: A/J, DBA/2J, 129X1/SvJ, and 129S1/SvImJ [9] . The data consist of 27.4 million capillary sequence reads giving a total of 5.3× coverage of the mouse genome. Sequences are from both ends of size-selected 2-, 10-, and 50-kbp clones derived from randomly sheared mouse genomic DNA. Second, the National Institute of Environmental Health Sciences contracted Perlegen Sciences to resequence by hybridization 15 inbred mouse strains [10] . This set includes 11 classical strains (129S1/SvImJ, A/J, AKR/J, BALB/cBy, C3H/HeJ, DBA/2J, FVB/NJ, NOD/LtJ, BTBR T+tf/J, KK/HlJ and NZW/LacJ) and four strains derived from the wild (WSB/EiJ, PWD/PhJ, CAST/EiJ and MOLF/EiJ), which represent the Mus musculus domesticus, Mus musculus musculus, Mus musculus castaneus and Mus musculus molossinus subspecies. Unlike the Celera resource, the hybridization approach used by Perlegen does not generate sequence reads and can only reliably detect single nucleotide polymorphisms (SNPs). Furthermore, the hybridization technology queried only 1.49 billion bases of the reference genome. This represents about 58% of the C57BL/6J sequence that is non-repetitive. The Perlegen approach was also found to have a false negative rate as high as 50% [8] . Therefore, currently available sequence data lack the coverage and breadth of strains to make a universal resource.

Here we present the sequence and analysis of mouse chromosome 17 from A/J and CAST/Ei generated using massively parallel sequencing and illustrate the power of this approach for the accurate and sensitive analysis of mouse genome variation. We selected mouse chromosome 17 for this sequencing project because at 95 Mb in length it is a shorter mouse autosome, and has a distinct cytogenetic profile that allows it to be easily and cleanly flow sorted. Biologically, mouse chromosome 17 is of great interest because the mouse major histocompatibility complex (MHC) resides on this chromosome [11] , as well as the murine t-complex, which in some wild-derived strains is responsible for transmission ratio distortion [12-14] . The strains A/J and CAST/Ei were selected for this pilot sequencing experiment because Celera has previously generated a significant number of shotgun capillary reads for A/J [9] , and while A/J is related to C57BL/6J, and these strains share several haplotype blocks on chromosome 17 [8] , CAST/Ei is highly divergent from the reference [8] . Indeed, the M. m. castaneus subspecific lineage is thought to have contributed less than 2% of the genomic material that makes up the classical laboratory strains of mice [8] . In addition, a large panel of congenic strains was derived from a consomic strain that has chromosome 17 from A/J substituted onto a C57BL/6J background [15] . These congenic strains have been extensively phenotyped to identify quantitative trait loci (QTLs) between C57BL/6J and A/J on chromosome 17. In particular, we were interested in using the A/J sequence of mouse chromosome 17 to refine a QTL reported to protect against high liver triglyceride levels [16] . We flow sorted mouse chromosome 17 from A/J and CAST/Ei and generated 22× and 34× coverage of these chromosomes, respectively. Using these data we identify and validate novel SNPs and structural variants between A/J, CAST/Ei, and the reference, and attempt a de novo assembly of these chromosomes. We illustrate that short read sequencing technology can be used to generate accurate assemblies of vertebrate experimental organisms such as mouse, allowing for the generation of a rich catalogue of variation between strains and the analysis of candidate genes within QTL intervals.
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Old 10-25-2010, 02:19 AM   #4
francois.sabot
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Check also Paux et al, Science (2007 ?). They have isolated the 3B chromosome of bread wheat (~800 Mb) and performed a BAC library with. They recently also made a NGS (solexa) analysis on this chromosome (at least a part of it).

Either, check fo Dolesel (?) paper of cyto isolation.

Cheers

Francois
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Old 10-25-2010, 02:16 PM   #5
Joann
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Plant J. 2004 Sep;39(6):960-8.

Dissecting large and complex genomes: flow sorting and BAC cloning of individual chromosomes from bread wheat.
Safár J, Bartos J, Janda J, Bellec A, Kubaláková M, Valárik M, Pateyron S, Weiserová J, Tusková R, Cíhalíková J, Vrána J, Simková H, Faivre-Rampant P, Sourdille P, Caboche M, Bernard M, Dolezel J, Chalhoub B.

Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Sokolovska 6, CZ-77200 Olomouc, Czech Republic.

Abstract
The analysis of the complex genome of common wheat (Triticum aestivum, 2n = 6x = 42, genome formula AABBDD) is hampered by its large size ( approximately 17 000 Mbp) and allohexaploid nature. In order to simplify its analysis, we developed a generic strategy for dissecting such large and complex genomes into individual chromosomes. Chromosome 3B was successfully sorted by flow cytometry and cloned into a bacterial artificial chromosome (BAC), using only 1.8 million chromosomes and an adapted protocol developed for this purpose. The BAC library (designated as TA-3B) consists of 67 968 clones with an average insert size of 103 kb. It represents 6.2 equivalents of chromosome 3B with 100% coverage and 90% specificity as confirmed by genetic markers. This method was validated using other chromosomes and its broad application and usefulness in facilitating wheat genome analysis were demonstrated by target characterization of the chromosome 3B structure through cytogenetic mapping. This report on the successful cloning of flow-sorted chromosomes into BACs marks the integration of flow cytogenetics and genomics and represents a great leap forward in genetics and genomic analysis.

PMID: 15341637 [PubMed - indexed for MEDLINE]
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