MIG-seq Library Preparation for Illumina Sequencing

The 1st PCR step was performed to amplify ISSR regions from genomic DNA with MIG-seq primer set-1 (Table 1). Alternatively, MIG-seq primer set-2 was used to create a different library from the same sample set (Supplementary Table 1). The volume of the PCR reaction mixture was 7 μl, containing 1 μl of template DNA, 0.2 μM of each 1st PCR primers, 3.5 μl of 2 × Multiplex PCR Buffer (Multiplex PCR Assay Kit Ver.2, Takara Bio, Kusatsu, Japan), and 0.035 μl of Multiplex PCR Enzyme Mix (Multiplex PCR Assay Kit Ver.2, Takara Bio). PCR was performed under the following conditions: initial activation at 94 °C for 1 min; 25 cycles for normal-concentration DNA (> 10 ng/μl) or 27 cycles for low-concentration DNA samples (< 5 ng/μl) from Calanoida copepod (see Supplementary Table 4) of denaturation at 94 °C for 30 s, annealing at 48 °C for 1 min and extension at 72 °C for 1 min; followed by a final incubation at 72 °C for 10 min, using a GeneAmp PCR System 9700 (Thermo Fisher Scientific). The PCR products were visualized using a Microchip Electrophoresis System (MultiNA; Shimadzu, Kyoto, Japan) with the DNA-2500 Reagent Kit (Shimadzu). Note that the 1st PCR could amplify a variety of ISSR regions, including some mismatched priming sites, depending on the conditions, because we decided to apply a relatively low annealing temperature (48 °C in our recommended system) for the 1st PCR after checking several different temperatures (data not shown). This annealing temperature could be effective to amplify more regions.
The 2nd PCR was performed to add the complementary sequences for the oligonucleotides that coat the Illumina sequencing flow cell, annealing sites of DNA sequencing primers, and indices to the 1st PCR products. The sequences of the common forward and indexed reverse primers were: 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCTG-3′ and 5′-CAAGCAGAAGACGGCATACGAGATxxxxxxGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC-3′, where “xxxxxx” denotes the six-base index (Supplementary Table 5). This PCR step was conducted independently to add individual indices to each sample using the common forward and indexed reverse primers. The six-base index was designed using the Barcode Generator by Luca Comai and Tyson Howell (http://comailab.genomecenter.ucdavis.edu/index.php/Barcode_generator). The 1st PCR product from each sample was diluted 50 times with deionized water and used as the template of the 2nd PCR. The 2nd PCR was performed in a 15-μl reaction mixture containing 3 μl of diluted 1st PCR product, 3 μl of 5 × PrimeSTAR GXL Buffer (Takara Bio), 200 μM of each dNTP, 0.375 U of PrimeSTAR GXL DNA Polymerase (Takara Bio), and 0.2 μM of common forward primer and individual reverse primer. The PCR conditions were as follows: 12 cycles of denaturation at 98 °C for 10 s, annealing at 54 °C for 15 s, and extension at 68 °C for 30 s. The concentrations of each 2nd PCR product (libraries) were measured using a Microchip Electrophoresis System (MultiNA, Shimadzu) with a DNA-2500 Reagent Kit (Shimadzu). The libraries from each sample, each with a different index, were then pooled in equimolar concentrations. To reduce the salt concentration, the mixed libraries were purified and the buffer was replaced with elution buffer using a QIAquick PCR Purification Kit (Qiagen, Venlo, Netherlands). Fragments in the size range of 300–800 bp in the purified library were isolated using Pippin Prep DNA size selection system (Sage Science, Beverly, MA, USA). The final concentration was measured using a SYBR green quantitative PCR assay (Library Quantification Kit; Clontech Laboratories, Mountain View, CA, USA) with primers specific to the Illumina system.