A total of 80 additional reactions were necessary to close gaps, to improve the genome sequence quality and to ensure correct assembly and to resolve any remaining base-conflicts. Assembly validation was Sunitinib c-Kit confirmed by pulsed-field gel electrophoresis (Figure 3) [2]. Figure 3 Pulsed field gel electrophoresis of Methanobrevibacter sp. AbM4 genomic DNA digested with restriction endonucleases (A) and in silico restriction enzyme maps (B). Panel A: Lane 1, undigested genomic DNA; Lane 2, digested with ApaI; Lane 3, digested with … Genome annotation A GAMOLA/ARTEMIS [31,32] software suite was used to manage genome annotation. Protein-encoding open reading frames (ORFs) were identified using the ORF-prediction program Glimmer [33] and BLASTX [34,35].

A manual inspection was performed to verify or, if necessary, redefine the start and stop codons of each ORF. Assignment of protein function to ORFs was performed manually using results from the following sources; BLASTP [34] to both a non-redundant protein database provided by the National Centre for Biotechnology Information (NCBI) [36] and clusters of orthologous groups (COG) database [37]. HMMER [38] was used to identify protein motifs to both the PFAM [39] and TIGRFAM [40] libraries. TMHMM [41,42] was used to predict transmembrane sequences, and SignalP, version 4.1 [43] was used for the prediction of signal peptides. Ribosomal RNA genes were detected on the basis of BLASTN searches to a custom GAMOLA ribosomal database. Transfer RNA genes were identified using tRNAscan-SE [44].

Miscellaneous-coding RNAs were identified using the Rfam database [45] utilizing the INFERNAL software package [46]. The AbM4 genome sequence was prepared for NCBI submission using Sequin [47]. The adenine residue of the start codon of the Cdc6-1 replication initiation protein (Abm4_0001) gene was chosen as the first base for the AbM4 genome. The nucleotide sequence of the Methanobrevibacter sp. AbM4 chromosome has been deposited in Genbank under accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”CP004050″,”term_id”:”509083678″,”term_text”:”CP004050″CP004050. Genome properties The genome of Methanobrevibacter sp. AbM4 consists of a single 1,998,189 bp circular chromosome Carfilzomib with an average G+C content of 29%. A total of 1,730 genes were predicted, 1,671 of which were protein-coding genes. A putative function was assigned to 1,258 of the protein-coding genes, while the remaining protein coding genes were annotated as hypothetical proteins. The properties and statistics of the genome are summarized in Tables 3 and and4,4, and are compared with genomes of other sequenced gut methanogens for the order Methanobacteriales in Table 5.