Precr repair mix

Given the anticipated DNA damage in the ancient permafrost samples, both iDNA and eDNA fractions were subject to DNA repair with PreCR™ Repair Mix (New England Biolabs, MA, USA) prior to sequencing. The PreCR™ Repair Mix is an enzyme cocktail (containing Taq DNA Ligase, Endonuclease IV, Bst DNA Polymerase, Fpg, Uracil-DNA Glycosylase, T4 Endonuclease V, and Endonuclease VIII) that can repair a wide range of DNA damages such as deaminated cytosine, apurinic/apyrimidinic sites, thymine dimers, nicks, and gaps. Up to 50 ng of DNA was treated with PreCR™ Repair Mix at 37 °C for 20 min according to the manufacturer’s instructions. The original untreated DNA, as well as the PreCR repaired DNA, were then converted to Illumina sequencing libraries using the Nextera DNA Flex Library Prep kit (Illumina, CA) with a unique DNA barcode added to each library. The libraries were examined on Bioanalyzer DNA High Sensitivity chips (Agilent, CA) for size distribution, and quantified by Qubit fluorometer (Invitrogen, CA). Each set of libraries were pooled at equal molar amount and sequenced on Illumina HiSeq 2500 Rapid flow cell as 2 × 150 nt paired-end reads. In total, 12 metagenomic libraries from the iDNA and eDNA fractions with and without DNA repair were sequenced. The pass-filter reads were retained and demultiplexed using fastq-multx for further analysis.

We selected the FFPE specimens by applying a multiplex PCR assay^{59 (link),60 (link)} to define the quality of the DNA extracted from the FFPE samples. This assay uses primer sets that amplify four genomic fragments (100, 200, 300, and 400 bp); the names and sequences of the primers are provided in Supplementary Table 3. Before the multiplex PCR assay, the DNA sample was heated at 90 °C for 60 min to facilitate the removal of the crosslinks of the FFPE tissues, and FFPE DNA repair was performed by adding 1× ThermoPol Buffer, 50x dNTPs/NAD + mixture, and PreCR Repair Mix (NEB) for 30 min at 37 °C^{57 (link)}. The samples producing 300- and 400-bp fragments were deemed to be good quality and were selected for this study. Ultimately, 29 cases of FFPE IMPC passed the quality control test.

DNA (15 µg) was treated with 5 ul of PreCR Repair Mix (New England Biolabs) in a 450 ul reaction in 1X ThermoPol buffer containing 0.1 mM (each) final concentration of dNTPs and 0.5mM final concentration of NAD+. DNA was then purified by one extraction with phenol/chloroform/isoamyl alcohol (25:24:1) and one extraction with chloroform followed by ethanol precipitation. 4 µg of PreCR-repaired DNA was used as input for the Nextera Mate Pair Sample Preparation kit (Illumina) following the manufacturer’s “gel plus” protocol. Size selection was performed with a BioRad FIGE Mapper using a buffer re-circulating pump and the following conditions: 1X TAE buffer; 16 hr run at room temperature; 4.1 V/cm forward and 2.7 V/cm reverse field strength, both with linear ramping from 0.1 sec initial to 0.8 sec final switch time. Gel slices were cut from the gel adjacent to ladder bands at ∼3kb, ∼6 kb and ∼10 kb. Circular ligation products were fragmented by sonication in a Diagenode Bioruptor NGS instrument (high power setting, 5 cycles of 15 sec on, 90 sec off). Fifteen cycles of enrichment amplification were performed.
Completed libraries were pooled as necessary and sequenced in paired-end Rapid Run mode on a HiSeq 2500 (Illumina). Read lengths were 151 bp forward read and 151 bp reverse read. Sequencing results for each library are given in Table S1.