Qsep100 dna fragment analyzer

We performed an MSI analysis on paraffin-embedded tissues to evaluate MSI status. The MSI status of the tumor samples was determined by using the five-marker Bethesda panel (BAT25, BAT26, D5S346, D2S123 and D17S250) [31 (link)]. Polymerase chain reaction (PCR) products were run on a Qsep 100 DNA fragment analyzer (Bioptic Inc., Taiwan, China) and analyzed using Qsep 100 viewer (Bioptic Inc., Taiwan, China). Microsatellite instability was defined by the presence of different sized alleles in tumor DNA compared with the matched normal DNA sample. We classified the results into microsatellite instability-high (MSI-H), microsatellite instability–low (MSI-L) and microsatellite stable (MSS) in tumors according to Bethesda guidelines [32 (link)].

We performed an MSI analysis on paraffin-embedded tissues to evaluate their MSI status. The MSI status of the tumor samples was determined using the five-marker Bethesda panel (BAT25, BAT26, D5S346, D2S123 and D17S250)^{59 (link)}. Polymerase chain reaction (PCR) products were run on an Qsep 100 DNA fragment analyzer (Bioptic Inc., Taiwan) and analyzed using Qsep 100 viewer (Bioptic Inc., Taiwan). Microsatellite instability was defined by the presence of different sized alleles in tumor DNA compared with the matched normal DNA sample. We classified the results into microsatellite instability-high (MSI-H), microsatellite instability–low (MSI-L) and microsatellite stable (MSS) in tumors according to Bethesda guidelines^{60 (link)}.

After amplification and purification, VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, Cat #ND607-01) was used to construct library. The input-DNA was quantified by Qubit2.0 (Life Technologies), and the size was measured with Qsep100™ DNA Fragment Analyzer (BIOptic). Then end-repair and adenylation were performed. The reaction mixture containing fragmented DNA (50 ng), end repair buffer, end repair enzymes, and nuclease-free water was incubated at 30 °C for 30 min and inactivated at 65 °C for 30 min. The finished end-prep reaction mixture was added with working adaptor and ligation enzymes and then was incubated at 20 °C for 15 min. The ligated DNA was purified, and size was selected with AMPure XP beads (Beckmen, Cat #A63881). The library amplification was followed, and purification was performed with AMPure XP beads. The final library was quantified by Qubit2.0 and the library size was measured with Qsep100™ DNA Fragment Analyzer. Library sequencing was performed using the NovaSeq 6000 and S4 Reagent Kit with paired-end reads of 150.

Microsatellites used in the study were selected from the microsatellite list recommended by ISAG/FAOs MoDAD (Measurement of Domestic Animal Diversity)
program (FAO, 2004). Microsatellites, their primer sequences and
chromosome numbers are given in Table 1. Annealing temperatures (ranged from
55.9 to 63.4  ${}^{\circ }$ C) for each locus were determined by gradient PCR method. Polymerase chain reactions were performed in a final volume of 20  $\mathrm{\mu }\mathrm{L}$ , including 10  $\mathrm{\mu }\mathrm{L}$ of 2X AmpMaster™ Taq (GenALL^®, South Korea), 1  $\mathrm{\mu }\mathrm{L}$ of (10  $\mathrm{pmol}\mathrm{\mu }{\mathrm{L}}^{-}$ ) each primers, 2  $\mathrm{\mu }\mathrm{L}$ of genomic DNA (30–50 ng) with ultrapure water added to the final volume. The selected microsatellite loci were amplified using a thermal cycler (Bio-Rad, USA). PCR mixture was heated to 95  ${}^{\circ }$ C for 5 min, followed by 35 cycles of, 30 s at a determined annealing temperature for each primer, with a final extension at 72  ${}^{\circ }$ C for 4 min. Amplified samples were controlled 2 % agarose gel, staining with EtBr (500  $\mathrm{\mu }\mathrm{L}{\mathrm{mL}}^{-\mathrm{1}}$ in ${\mathrm{H}}_{\mathrm{2}}\mathrm{O}$ ). The microsatellite loci studied in the study were genotyped by capillary electrophoresis in Qsep 100™ DNA fragment analyzer (BiOptic Inc., USA) without fluorescently labeled primers. The obtained results were evaluated using
BiOptic software provided by the manufacturer.