Physarum polycephalum Sequencing Plan

Introduction

The Physarum polycephalum genome has been targeted for a high quality draft and assembly by the NHGRI. An examination of available data available from the community and our preliminary results indicate that the ~300 Mb genome is about 40% repetitive, including about 100 families of 2000 members that are relics of transposition events. There are many repetitive elements in the intergenic regions, and there are homopolymer sequences in the introns of many genes. They are composed of poly A or poly C stretches of 10-25 nt on the same strand. From analysis of the genome survey sequence, it has been determined that there are two transoposon types, an 8.3 kb LTR-retrotransposon-like sequence (TP1) that has inserted into itself, generating 20-50-kb islands of repeated, hypermethylated DNA, and another LTR-retrotransposon-like sequence of 1.68 kb (TP2). These repeats may confound the assembly process, but we feel that an initial attempt at 6X coverage, along with EST data, will give us ample information on which to formulate alternatives, if necessary.

Gernot Gloeckner and Wofgang Marwan, Physarum community researchers, plan to produce a map with markers spaced every 100 kb. The map is to be produced in parallel to the sequencing effort, and is to be integrated with the sequence. In addition, the community has cDNA resources available that can be used for the sequencing effort. The community has already determined that the available Dictyostelium genome sequence will be of great use in annotating the Physarum sequence (Gerard Pierrion, personal communication).

Sequencing Plan

This initial plan was developed in consultation with and using data input from members of the community as Jonatha Gott, Gerard Pierrion, Gernot Gloeckner, Wofgang Marwan and others. The goal is to provide resources that will be useful to the community, taking into consideration the anticipated difficulties of the genome and relative cost effectiveness of the project.

DNA is in house and was prepared from haploid amoebe provided by the laboratory of Gerard Pierrion. We propose to perform a 6X WGS with plasmid (5.7X) and fosmid end sequences (0.3X). We also propose to perform cDNA sequencing on the 454 FLX Sequencer, obtaining as much full length sequence as possible, and to use community cDNA library resources for EST production. The whole genome data will be assembled, and we will evaluate the genome, perhaps returning at a later date with additional requests, armed with additional data. If it appears that a standard 6X WGS will not assemble well, options might include:

determining a Cot curve and sampling the high Cot fraction to produce a Physarum-specific repeat database
sequencing the low cot fraction to determine the unique regions
preparing fosmid filters for hybridization to identify and sequence clones of interest to the community