Hifi pcr kit

The lentiviral mouse Smn1 expressing vector activated by EF1 alpha promoter was constructed from the pSin-EF2 plasmid (#16578, addgene, Watertown, MA, USA) by replacing NANOG gene with SpeI and BamHI. Human SMN1 carrying flag sequence was replaced into the same plasmid through SpeI and EcoRI cutting site. Inserted genes were amplified from cDNA of mouse testis or hciPSCs by HiFi PCR Kit (KR0368, KAPA Biosystems, Wilmington, MA, USA) using specific primers listed in Supplementary Table S1. Packaging and envelop vectors from RNAi core (Academia Sinica, Taipei, Taiwan) were transfected together with lentiviral expressing vector into 293T cells to produce Flag-hSMN1 lentivirus. TrypLE express-dissociated single hiPSCs were infected in suspension with SMN-lentivirus for one day in Metrigel coated plates. On the second day, the virus was removed and changed to fresh StemFlex medium until colony regrows to 0.5 mm diameter.

DNA from stool samples was extracted with the Qiagen DNA Stool kit following the manufacturer’s instructions. Two steps of Polymerase Chain Reaction (PCR) procedures were used to generate amplicons from the 16S RNA genes for sequencing. The first-round PCR was to target V3/V4 regions of 16S rRNA genes with the forward primer: 5′- CCTACGGGNGGCWGCAG and the reverse primer: 5′- GACTACHVGGGTATCTAATCC. This step was done with the KAPA Biosciences HiFi PCR kit and additional BSA. The protocol consists of initial denaturation at 95 °C for 3 min, followed by 25 cycles of denaturation (90 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s), and final elongation at 72 °C for 5 min. The PCR products were cleaned up with Ampure XP beads. The second-round PCR was performed with Nextera XT index Primers and sequencing Adaptors with the following setting: initial denaturation at 95 °C for 3 min, followed by 8 cycles of denaturation (90 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s), and final elongation at 72 °C for 5 min. The PCR products were cleaned up with Ampure XP beads and paired-end sequenced (2 × 300 bp) on an Illumina MiSeq platform at the University of North Carolina at Charlotte.

Our approach relies on a single COI probe (MCSP: Metabacording by Capture with a Single Probe). We chose a COI probe (hereafter named single-probe) from a species (Danio rerio) belonging to an order (Cypriniformes) absent from our study area, the Amazon basin.
The single-probe was developed by PCR amplification of the CO subunit 1. PCR was performed with 80 ng D. rerio DNA, 0.3mM of forward and reverse [5']-Biotin-TEG primers [23 (link)] (FishF = TCA-ACC-AAC-CAC-AAA-GAC-ATT-GGC-AC, FishR = TAG-ACT-TCT-GGG-TGG-CCA-AAG-AAT-CA), 12.5 μl HiFi PCR kit (KR0369, KAPA Biosystem), 9 μl H2O. PCR was run with the following cycling protocol: 95–3’, 98–80”, 52–40”, 72–1’- 35 cycles [24 ].The PCR product (682 bp) was purified with 1X ampure XP and then quantified.
In this study, we will compare the result from this new approach to a previous approach using four probes designed for siluriforms [21 (link)]. In the following text, we will use the term single-probe for the COI from Danio and the term siluriform probes for the four probes previously developed [21 (link)].

The sequences of primers used for pyrosequencing are listed in Additional file 1: Table 1. After amplification using the HiFi PCR Kit (KAPA), the PCR products were combined with the reaction binding beads and placed in a pyrosequencing detector (PyroMark q96 ID, Qiagen) for the reaction. Sequencing data were analyzed using the Pyro Q-CpG software.