Three serendipitous pathways in E. coli can bypass a block in pyridoxal-5′-phosphate synthesis

Overexpression of seven different genes restores growth of a ΔpdxB strain of E. coli, which cannot make pyridoxal phosphate (PLP), on M9/glucose.None of the enzymes encoded by these genes has a promiscuous 4-phosphoerythronate dehydrogenase activity that can replace the activity of PdxB.Overexpression of these genes restores PLP synthesis by three different serendipitous pathways that feed into the normal PLP synthesis pathway downstream of the blocked step.Reactions in one of these pathways are catalyzed by low-level activities of enzymes of unknown function and a promiscuous activity of an enzyme that normally has a role in another pathway; one reaction appears to be non-enzymatic

Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction

Background Ribosomal RNA (rRNA) comprises at least 90% of total RNA extracted from mammalian tissue or cell line samples. Informative transcriptional profiling using massively parallel sequencing technologies requires either enrichment of mature poly-adenylated transcripts or targeted depletion of the rRNA fraction. The latter method is of particular interest because it is compatible with degraded samples such as those extracted from FFPE and also captures transcripts that are not poly-adenylated such as some non-coding RNAs. Here we provide a cross-site study that evaluates the performance of ribosomal RNA removal kits from Illumina, Takara/Clontech, Kapa Biosystems, Lexogen, New England Biolabs and Qiagen on intact and degraded RNA samples. Results We find that all of the kits are capable of performing significant ribosomal depletion, though there are differences in their ease of use. All kits were able to remove ribosomal RNA to below 20% with intact RNA and identify ~ 14,000 protein coding genes from the Universal Human Reference RNA sample at >1FPKM. Analysis of differentially detected genes between kits suggests that transcript length may be a key factor in library production efficiency. Conclusions These results provide a roadmap for labs on the strengths of each of these methods and how best to utilize them. Keywords: RNAseqr; RNA depletion; Illumina; NGS; ABRF; TranscriptomicsNational Cancer Institute (U.S.) (Grant P30-CA14051)National Institute of Environmental Health Sciences (Grant P30-ES002109