DNA Extraction, PCR, Cloning and Sequencing
Genomic DNA was extracted from a maximum of 1.0 g of soil using the
MoBio Powermax kit following manufacturer’s instructions. PCR of soil
DNA extracts utilized Redtaq (0.25 units/rxn) (Sigma-Aldrich Corp. St.
Louis, MO), and primers FGPS989ac (GGGGTCCGTAAGGGTC) (Bosco et
al . 1992) and 23SFr3-ten (GGCAWGGGTGACAGGATTTA), the latter a
degenerate version of a primer we developed for this study. This primer
set targets the non-coding intergenic spacer region (IGS) between the
16S and 23S ribosomal RNA loci. Cycling conditions were: initial
denaturation at 94°C for 4:00 min, 35 cycles of 94°C for 1:00 min,
annealing for 0:30 secs (55°C for FGPS989ac/23SFr3-ten), 72°C for 1:30
min, and finally 72°C for 5:00 min. Amplicons from soil DNA extracts
were cloned into the TOPO TA 4.0 vector with chemically competent Top10
cells following the manufacturer’s instructions (Invitrogen, Carlsbad,
CA). Individual colonies (100 per library) were selected at random from
each plate for PCR amplification using M13 forward and reverse primers,
and sequencing of the insert, which was performed at Functional
Biosciences (Madison, WI). We opted to clone our PCR products because
the high GC content of the target locus interferes with sequencing. We
were able to obtain much cleaner reads using the M13 vector primers. We
hypothesize that the cloning and Sanger sequencing approach used here
should result in less bias (particularly against high GC targets) than a
direct NGS approach, and also provides longer contiguous sequences for
analysis than the NGS methods available at the time of our benchwork.
DNA sequences were edited using CodonCode Aligner (CodonCode
Corporation, Centerville, MA), and multiple sequence alignments were
generated using the Muscle (Edgar, 2004) and Mafft (Katoh et al .,
2002) programs on the CIPRES (Miller et al . 2010) network.
Sequence alignments were corrected by eye in Seaview (Gouy et
al ., 2010) and trimmed to common length using Bioedit (Hall 1999).
Likely chimeric sequences were identified using the program UCHIME
(Edgar et al ., 2011) as implemented in the mothur package
(Schloss et al ., 2009), and removed prior to subsequent analysis.
Sequences derived from our mid-succession control plots were
consistently shorter than those from any other clone library. We
therefore generated two alignments for analysis: one that spanned the
entire rIGS but excluded mid-succession control sequences (811 bp final
alignment), and one that was truncated within the rIGS region and that
only included sequences from mid-succession libraries (627 bp final
alignment). We used the former as the basis for our phylogenetic
analysis and comparison of early and late succession libraries and,
within the latter, of alder and non-alder soils. The mid-succession-only
alignment was used to compare differences among fertilization treatments
in our mid-succession sites.