Figure 2
From: Building de novo reference genome assemblies of complex eukaryotic microorganisms from single nuclei
From a soil sample to AM fungal genome assemblies. (a) Whole inoculum from the culture collection INVAM is blended with water and (b) poured into a set of sieves; the material stuck in the 38 μm sieve is placed into a (c) tube that contains a solution of 60% sucrose, then centrifuged for 1 min. The supernatant is run through a 38 μm sieve and washed with water. (d) The sieve content is placed in a Petri dish for the spores to be manually picked using a glass pipette. (e) After cleaning the spores with ddH2O, these are placed one-by-one into tubes and crushed with a pestle. (f) The DNA from a broken spore is stained with SYBR Green, giving a strong fluorescent signal for the nuclei and a lighter signal for the background, organelles and microbes. (g) The stained spore content is loaded on the FACS, where the sample moves inside a constant flow of buffer and crosses a laser beam. An excitation laser of 488 nm and 530/40 band pass filter was used for the SYBR Green fluorescence detection. In addition, scattered light, forward scatter (FSC) and side scatter (SSC) were used as proxy for size and granularity to identify the nuclei. (h) The signals can be interpreted in a scatterplot, and particles of a selected cloud (e.g., R1, blue-box) can be sorted individually or pooled (i) into individual wells of a 96-well plate by directing them with a charge. (j) The content of each well is whole genome amplified using MDA. (k) The amplified products are tested for fungi and bacteria by PCR screening with specific rDNA primers. The products confirmed to be from fungal nuclei are sequenced with (l) Illumina HiSeqX, for single nuclei; and (m) Oxford Nanopore, for pools of nuclei. (n) In workflow 1, Illumina reads are assembled separately for individual nuclei using MaSuRCA39. (o) In workflow 2, reads from individual nuclei are normalized and assembled with SPADES40. (q) In workflow 3 reads from all nuclei are combined, then normalized and finally assembled with SPADES40. (p) Lingon38 is used to produce a consensus assembly from individual nuclei assemblies in both workflows 1 and 2. (r) Nanopore data is assembled with Canu41, polished with Pilon53 using the Illumina raw-reads and used to (s) scaffold the three assemblies generated with workflows 1, 2 and 3 using Chromosemble, of Satsuma55.
