【正文】 Mitochonrium and chloroplast DNA enriched libraries 96 wells capillary high throughput automatic seqencing Sequencing line? Platform? from colony to read, w/o hand operation within our imaging ability quality (base calling standard) vector trimming (searching and deleting) trim length 100bp excluded trim length 100bp “passing” among passing shotgun sequences, repeats kept providing redundant coverage clone coverage, the span of genome covered by passing clones Weight, the fraction of total passing reads from the specified library sequence and clone depth, the total span of passing clones dividing by the estimated total genome size (34 Mb) Polymorphism rate in T. Pseudonana Thin black line: fraction of nucleotides as a function of coverage Dashed black line: fraction of nucleotide positions as a function of coverage with two or more reads Thick black line: fraction of polymorphic positions as a function of coverage Polymorphisms between the two haplotypes (Asexually propagated or mitotic pure population) are estimated to occur at % of the nucleotide positions based on parison of multiple overlapping reads at each site, and requiring two or more matches to two bases at a position to call a position polymorphic Reads passing the primary quality and vector screens (passing reads) were assembled into scaffolds by means of JAZZ, a modular suite of tools for large shotgun assemblies that incorporates both readoverlap and readpairing information Overlapping with allowed variation + linking information A file containing these reads in FASTA format () can be downloaded from the JGI’s Thalassiosira pseudonana web portal The unified consensus sequence is a mosaic of the two haplotypes The assembly consists of 2,170 contiguously assembled segments (contigs) longer than 2 kb with a total length of million base pairs (Mb) linked by pairedend constraints into 1271 scaffolds spanning Mb Half the assembled nucleotides are in 36 contigs longer than 246 kb and 19 scaffolds longer than Mb Quality and quality assessment Assembly of Organellar Genomes Screening the whole genome sequence (WGS) reads for anellar sequences for the known plastid and mitochondrial genomes, finding a large set (2117) of candidate reads Sequencing a library of anellarenriched DNA provided an additional 8,428 reads. All were assembled with the Phred Phrap package, resulting in single contig assemblies for both anellar genomes. Plastid assembly was inplete due to the inverted repeat (IR) mon in chloroplast genomes. Using consed ( a region was identified with a sharp increase in coverage, a signal of the IR. The reads assembling into the IR and 4 conjoined short single copy (SSC) regions were separated and assembled separately. The resulting contig was blasted to the original assembly and the second IR copy was pasted onto the end of the original assembly As verification, the assembly was electronically digested with the restriction enzyme NheI and pared it to the plastid genome optical NheI restriction maps The assembly fragments aligned to the optical map within allowed tolerances. For additional confidence, PCR primers were designed to span the manually joined region, from the 539。 end of the SSC into the IR, and from the large single copy (LSC) region into the IR. All primer pairs generated the expected PCR fragment sizes, and confirmed our assembly technique as well as the circularity of the plastid genome Optical restriction site mapping A) Genomic DNA is extracted, elongated and immobilized onto a positively charged glass surface through a bination of micro?uidics and electrostatic interactions B) A restriction enzyme cleaves the surfacebound molecules, which are then stained with a ?uorescent dye C) The digested single DNA molecules are imaged by a fully automated ?uorescence microscopy scanning system that rapidly creates highresolution images D) Using the integrated ?uorescence intensity measurements of the imaged restriction fragments, machine vision calculates the mass of each fragment E) Ordered restriction maps, each representing a single DNA molecule, are assembled into contigs with consensus maps that span entire genomes ChannelCollect Gentig from enough and qualified (Read) raw data on Overlapping + Linking principle by powerful Softwares, Computer and sometime References ROLSCaR strategy (methodology) Oscar Prize (award) 某 女 Schematic representation of 24 chromosomes, including 9 with two haplotypes (a or b). Blue lines, optical maps of chromosomes, and goldcolored lines, wholegenome scaffolds with gaps. R, reverse plementation. Scaffolds assigned to the map based on parison between the map and in silico scaffold digests are positioned adjacent to the matching segment of the map. Scaffolds that cannot be unambiguously placed on the mapareshowninthe box. Gene modeling (digging based on typical characters) cross words word puzzle picture recovering from 1000 pieces and clustering (function related groups) annotation? Function of known gene sequence one by one group by group characterization? Structure of known sequence finding + identification Prior to this project, few fulllength genes had been described for T. pseudonana (only 8 nonribosomal genes) or for diatoms in general. Our gene prediction method therefore relied heavily on homology to known sequences from other anisms 有沒有已經(jīng)知道的蛋白質(zhì) We first used BLASTX to align all proteins in GenBank (release 131) to the T. pseudonana assembly in translation (1 of 3 possibilities) BLAST parameters: expect value cutoff e–