Genome sequencing of the Lactobacillus fabifermentans T30PCM01 isolated from grape marcs
Growth of cells and DNA preparation.  For the isolation of L. Fabifermentans chromosomal DNA, an overnight culture was grown in in 100 ml of M-17 broth at 37°C. Cells were harvested by centrifugation and the pellet was resuspended in 10 ml of a TE solution (10 mM Tris-hydrochloride, 1 mM EDTA,pH 7.5) containing lysozyme (0.5 mg/ml). The suspension was incubated at 37°C for 30 min. Afterwards the cells were collected and resuspended in 4 ml of TE (50 mM Tris-hydrochloride, 20 mM EDTA, pH 8) containing 1% SDS. The suspension was mix gently by intermittent inversion and, to ensure complete lysis, was kept at 65°C for 20 min. Afterwards 2 ml of KAc 5 M were added to the lysate and the solution was maintained at 0°C for 30 min. After centrifuging at 12000xg for 30 minutes at 4°C the supernatant was recovered and DNA was precipitated using ethanol. The pellet was air-dried and resuspended in 1 ml of TE (10 mM). Sample was treated with RNAse A (0.2 mg/ml) for 30 min at 37°C and treated with proteinase K (0.3 mg/ml) for 30min at 56°C. DNA was precipitated by adding an isovolume of isopropanol and pelleted by centrifugation. Pellet was air-dried and resuspended in nuclease free water.Finally the DNA was purified using phenol-chloroform. Genomic DNA quantification and purity were determined using Nanodrop and Qubit. Absence of degradation was verified with agarose gel electrophoresis using a 0.8% gel.  Genomic DNA was sequenced at the Ramaciotti Centre for Gene Function Analysis (UNSW-Sydney) using the Illumina MiSeq Benchtop Sequencer. Sequencing produced 1413271 paired-end reads of 250+250 basi. Lybraries were produced using the “Nextera XT” kit, the DNA insert size was between ~350 bp up to 1.5 kbp. Sequence quality and conversion to fastaq format was performed using the FASTX-Toolkit version 0.0.13. Assembly was performed both on the quality trimmed and quality-filtered sequences and on the original sequences. The best result in terms of N50, maximum scaffold size and number of scaffolds were obtained on the original (not-filtered) sequences. End-trimming was performed using quality threshold “13” and quality filtering was performed selecting only the sequences having less that 10% of the sequence with quality lower than 13. Finally all the sequences shorter than k-mer length used for assembly were discarded.  Short reads were assembled using the ABySS software (ver. 1.3.6) with the shortPaired procedure and a kmer of 63. Assembly generated 93 scaffolds larger than 1000 bases, the remaining scaffold were discarded.