Combinatorial Pooling and Resequencing

[abaten]

Just sharing a NEXT-Gen issue for your suggestions.
Currently we have 4000 BACs and we would like to sequence them.
However, to reduce the time and cost of current barcoding scheme, we are interested in knowing/applying any optimal pooling scheme which may reduce the number of barcodes to be used. The objective is-
1) Nnstead of using 4000 barcodes for 4000 specimens, they can be grouped into different pools (only several hundred pools, each pool containing several specimens) and barcode individual pools, rather than individual specimens.
2) Once the pooling and the sequencing is done, retrieve the sequence identity (i.e. Which sequence came from which specimen)

In this regard, we are looking for public domain techniques/protocols (public domain, which we will be able to use) to employ for the above mentioned problem.

Thanks in advance for your suggestions.

[krobison]

I think BACs raise the ante a few orders of magnitude in terms of challenge in decoding, but Chang et al below would seem to be attacking a similar problem.

Presumably after you pooled with their scheme and sequenced, the first step would be to eliminate purely vector sequences. Running remaining sequences for each pool through velvet (or similar) would then give a set of contigs, which when compared against each other could be used to decompose the pools based on unique sequences (repetitive stuff will probably be pain). That decomposition could be used to drive a new round of assembly.

Genome Res. 2009 Jul;19(7):1243-53. Epub 2009 May 15.
DNA Sudoku--harnessing high-throughput sequencing for multiplexed specimen analysis.
Erlich Y, Chang K, Gordon A, Ronen R, Navon O, Rooks M, Hannon GJ.

Watson School of Biological Sciences, Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.
Next-generation sequencers have sufficient power to analyze simultaneously DNAs from many different specimens, a practice known as multiplexing. Such schemes rely on the ability to associate each sequence read with the specimen from which it was derived. The current practice of appending molecular barcodes prior to pooling is practical for parallel analysis of up to many dozen samples. Here, we report a strategy that permits simultaneous analysis of tens of thousands of specimens. Our approach relies on the use of combinatorial pooling strategies in which pools rather than individual specimens are assigned barcodes. Thus, the identity of each specimen is encoded within the pooling pattern rather than by its association with a particular sequence tag. Decoding the pattern allows the sequence of an original specimen to be inferred with high confidence. We verified the ability of our encoding and decoding strategies to accurately report the sequence of individual samples within a large number of mixed specimens in two ways. First, we simulated data both from a clone library and from a human population in which a sequence variant associated with cystic fibrosis was present. Second, we actually pooled, sequenced, and decoded identities within two sets of 40,000 bacterial clones comprising approximately 20,000 different artificial microRNAs targeting Arabidopsis or human genes. We achieved greater than 97% accuracy in these trials. The strategies reported here can be applied to a wide variety of biological problems, including the determination of genotypic variation within large populations of individuals.

PMID: 19447965


You might also look at the below two papers; perhaps there is a sequencing strategy related to these that would help. For example, perhaps the Young et al strategy would work with relatively complex pools of BACs. It would be really clever if the Hiatt et al strategy could be specifically directed at the BAC-vector junctions, as this would fingerprint each BAC within your pooling strategy. Or, if in a similar way you could create mate pair libraries where one mate was always a BAC-vector junction but the distance between mates varied widely -- that way a large number of tags could be unambiguously assigned to a given clone & used to help corral other fragments into the right bin.

Genome Res. 2010 Feb;20(2):249-56.
A new strategy for genome assembly using short sequence reads and reduced representation libraries.
Young AL, Abaan HO, Zerbino D, Mullikin JC, Birney E, Margulies EH.

Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
We have developed a novel approach for using massively parallel short-read sequencing to generate fast and inexpensive de novo genomic assemblies comparable to those generated by capillary-based methods. The ultrashort (<100 base) sequences generated by this technology pose specific biological and computational challenges for de novo assembly of large genomes. To account for this, we devised a method for experimentally partitioning the genome using reduced representation (RR) libraries prior to assembly. We use two restriction enzymes independently to create a series of overlapping fragment libraries, each containing a tractable subset of the genome. Together, these libraries allow us to reassemble the entire genome without the need of a reference sequence. As proof of concept, we applied this approach to sequence and assembled the majority of the 125-Mb Drosophila melanogaster genome. We subsequently demonstrate the accuracy of our assembly method with meaningful comparisons against the current available D. melanogaster reference genome (dm3). The ease of assembly and accuracy for comparative genomics suggest that our approach will scale to future mammalian genome-sequencing efforts, saving both time and money without sacrificing quality.

PMID: 20123915


Nat Methods. 2010 Feb;7(2):119-22. Epub 2010 Jan 17.
Parallel, tag-directed assembly of locally derived short sequence reads.
Hiatt JB, Patwardhan RP, Turner EH, Lee C, Shendure J.

Department of Genome Sciences, University of Washington, Seattle, Washington, USA. [email protected]
Comment in:

Nat Methods. 2010 Feb;7(2):105-6.
We demonstrate subassembly, an in vitro library construction method that extends the utility of short-read sequencing platforms to applications requiring long, accurate reads. A long DNA fragment library is converted to a population of nested sublibraries, and a tag sequence directs grouping of short reads derived from the same long fragment, enabling localized assembly of long fragment sequences. Subassembly may facilitate accurate de novo genome assembly and metagenome sequencing.

[[email protected]]

Reduced representation library protocol

Hi All,

Over the coming weeks I am going to produce some RRL to sequence on the solexa GAIIx platform. I have made some RNAseq libraries previously but not RRL. Does anyone have a working protocol? I have the enzymes selected for my species but am unsure of the volume of starting material required and the sequence of clean up steps for the highest quality sequence. Have read many recent publications but as usual the methods are brief.

Thanks in advance for your help.