Coastal saltmarsh sediments represent an important source of natural methane emissions, much of the methanogenesis originates from quaternary and methylated amines, such as choline and trimethylamine. However, the key microbes involved in choline-dependent methanogenesis remain poorly characterized and the metabolic pathways by which the saltmarsh microbes degrade choline and form methane are yet to be determined. In this study, we combined DNA stable isotope probing microcosms with high throughput sequencing of 16S rRNA genes and 13C2-choline enriched metagenomes, followed by binning of the microbial population genomes to identify the microbes responsible for methanogenesis. Microcosm incubation with 13C2-choline leads to the formation of trimethylamine and subsequent methane production, suggesting that choline-dependent methanogenesis is a two-step process involving trimethylamine as the key intermediate. Amplicon sequencing analysis identified Deltaproteobacteria, of the genera Pelobacter and Desulfuromonas were the major choline-utilizers. The methanogenic Archaea, of the genera Methanococcoides became enriched in choline-amended microcosms, indicating their role in methane formation from trimethylamine. The binning of the microbial population genomes from metagenomic DNA resulted in the identification of bins that are classified as Pelobacter, Desulfuromonas, Methanococcoides and their associated viruses. Analyses of these bins revealed that Pelobacter and Desulfuromonas have the genetic potential to degrade choline to trimethylamine using the choline-trimethylamine lyase pathway, whereas Methanococcoides are capable of methanogenesis using the pyrrolysine-containing trimethylamine methyltransferase pathway. Due to issues with the ENA submission process I have not split my samples, so they still contain barcodes: T0.A.F12.12C.EJ CACTACGCTAGA T0.B.F12.12C.EJ TACTACGTGGCC T0.C.F12.12C.EJ CGGTCAATTGAC T0.A.F7.12C.EJ TCCCTTGTCTCC T0.B.F7.12C.EJ TGCATACACTGG T0.C.F7.12C.EJ GCGATATATCGC T1.B.F12.12C.EJ AGTCGAACGAGG T1.D.F12.12C.EJ CGAGCAATCCTA T1.I.F12.12C.EJ TGCAGTCCTCGA T1.I.F7.12C.EJ ACGAGACTGATT T1.B.F7.12C.EJ GGTGACTAGTTC T1.D.F7.12C.EJ CGTAAGATGCCT T1.B.F12.13C.EJ TCGACATCTCTT T1.I.F12.13C.EJ CTATCTCCTGTC T1.L.F12.13C.EJ AGGCTTACGTGT T1.B.F7.13C.EJ ATCACCAGGTGT T1.I.F7.13C.EJ GAATACCAAGTC T1.L.F7.13C.EJ GTATCTGCGCGT T2.E.F12.12C.EJ GCTGTACGGATT T2.G.F12.12C.EJ ACCAGTGACTCA T2.C.F12.12C.EJ GCGTTCTAGCTG T2.C.F7.12C.EJ GGCCAGTTCCTA T2.E.F7.12C.EJ GTGGAGTCTCAT T2.G.F7.12C.EJ ATGGGTTCCGTC T2.F.F12.13C.EJ GTAGATCGTGTA T2.J.F12.13C.EJ CGAGGGAAAGTC T2.K.F12.13C.EJ GAACACTTTGGA T2.K.F7.13C.EJ TGGTCAACGATA T2.F.F7.13C.EJ AACTAGTTCAGG T2.J.F7.13C.EJ GGACTTCCAGCT T3.F.F12.12C.EJ GCTCGAAGATTC T3.J.F12.12C.EJ TAGGCATGCTTG T3.K.F12.12C.EJ GTTGTTCTGGGA T3.F.F7.12C.EJ AGTCGTGCACAT T3.J.F7.12C.EJ ACCATAGCTCCG T3.K.F7.12C.EJ GATGTTCGCTAG T3.G.F12.13C.EJ ATCGCACAGTAA T3.H.F12.13C.EJ TAACGTGTGTGC T3.D.F12.13C.EJ CTCACAACCGTG T3.G.F3.13C.EJ CAAATTCGGGAT T3.D.F7.13C.EJ ACTCACAGGAAT T3.G.F7.13C.EJ TCTCTACCACTC T3.H.F7.13C.EJ ATTCTGCCGAAG Linker: GTGCCAGCMGCCGCGGTAA The mapped file sample names are in this format: T0.A.F12.12C.EJ The parts of the sample names refer to: T0 is the timepoint A is the replicate number (T0 technical triplicates, T1,2,3 biological triplicates) F12 is the fraction (F12 = light, F7 = heavy) 12C is the form of choline the microcosm was enriched with (NB T0 samples are actually unamended, so no choline was actually added)