Truseq nano

For 6 selected samples, from field, day 0, and day 50 for the bog and fen, metagenome sequencing was also performed using 100 ng of the extracted DNA in TruSeq (Illumina) library preparations (field samples) or TruSeq Nano (Illumina) library preparations (incubation samples), using an Illumina NextSeq500. The small sample size of these metagenomes precluded statistically meaningful comparisons of the overall community, so analyses focused on the methanogens.

Yeast genomic DNA was extracted using the spheroplast method (http://fangman-brewergeneticswashingtonedu/indexhtml). Samples were prepared according to the TruSeq Nano sample preparation guide from Illumina. To generate replication timing profiles, the ratio of uniquely mapped reads in the replicating samples to the nonreplicating samples was calculated following Müller et al. (2014) (link), and profiles were smoothed by a Fourier transformation (Müller et al. 2014 (link)). A replication peak was defined as a curve point where the S to G1 ratio/Δkb changed from plus to minus and the same sign was kept at more than 3 kb from the change point. A peak is therefore defined as a local maximum. The values of T_rep were from OriDB (Siow et al. 2012 (link)).

Microsatellite sequences from O. vulgare L. ssp. hirtum were identified as a service by Ecogenics GmbH following NGS sequencing of an O. vulgare L. ssp. hirtum genomic DNA sample. The Illumina TruSeq nano DNA library was sequenced on an Illumina MiSeq sequencing platform using a nano v2 500 cycles sequencing chip. The resulting paired-end reads which passed Illumina’s chastity filter were subjected to de-multiplexing and trimming of Illumina adaptor residuals. Subsequently the quality of the surviving reads was checked with FastQC v0.11.8 [14 ]. In a next step, the paired-end reads were quality filtered and merged with USEARCH v11.0.667 [15 (link)] to in silico reform the sequenced molecules. The resulting merged reads were screened with the software Tandem Repeats Finder, v4.09 [16 (link)]. After this process, 6121 merged reads contained a microsatellite insert with a tetra- or a trinucleotide of at least six repeat units or a dinucleotide of at least ten repeat units. Primer design was performed with primer 3 [17 (link),18 (link)]. Raw NGS sequences can be accessed at the NCBI Sequence Read Archive under project number PRJNA921701.

DNA was isolated from fresh-frozen tissue, using AllPrep DNA-RNA-miRNA Universal Kit (QIAGEN #80224). 108 Illumina TruSeq Nano libraries were prepared from 100 ng DNA each according to protocol. Hybridization capture was performed using the IDT xGen Pan-Cancer panel v1.5, which targets cancer genes identified by The Cancer Genome Atlas (TCGA). Sequencing was performed on Illumina NextSeq, using 2 × 150 bp paired-end reads. We have securely archived the fastq sequencing files at the European Genome-Phenome Archive (EGA) under study accession ID EGAS00001004108. The EGA is subject to the EU’s General Data Protection Regulation and access to the data may be applied for, subject to a data-access agreement with project description and ethics board approval.

DNA was extracted from a silica-dried leaf tissue sample from a cata-morph individual collected from Pakke Tiger Reserve, using a modified CTAB protocol at TrEE Lab, IISER Bhopal [45 ]. Eight such extractions were performed using leaf tissue from the same individual followed by gel extraction using a QIAEX II kit (Qiagen, Valencia, CA, USA) following the manufacturer’s protocol. The DNA from all the samples was pooled and purified using Genomic Tips kit (Qiagen, Valencia, CA, USA). The sample was then concentrated using a Savant SpeedVac (Thermo Fisher Scientific, Waltham, MA, USA). An Illumina Tru-Seq Nano gel-free library with approximately 550 bp insert size was prepared from the DNA sample. The genome was finally sequenced at Edinburgh Genomics on a single lane of an SP flowcell on the NovaSeq 6000 (Illumina, San Diego, CA, USA) with 250 bp paired-end reads, with the aim of generating 100x sequencing coverage.

Methods for DNA extraction from and sequencing of the 214 bulk soil metagenomes are as previously described^{14 ,31 (link)}. Briefly (with a few exceptions¹⁴ ), 100 ng DNA per sample was used for TruSeq Nano (Illumina) library construction. The 2012 libraries yielded 100-bp paired-end reads from 1/12th of an Illumina HiSeq2000 lane, and the 2011 libraries yielded 150-bp paired-end reads from 1/24th of an Illumina NextSeq lane.