TITLE: Whole-mitogenome sequencing of Oncorhynchus masou masou by next-generation sequencing
AUTHORS: Yoko Kato-Unoki, Kosuke Tashiro

[STEPS]
?. [Preparation of the mitogenome libraries for next-generation sequencing]
Amplify and purify the mitogenome libraries by using a QIAseq FX DNA Library Kit (QIAGEN) and Agencourt AMpure XP beads (Beckman Coulter), respectively. To reduce reagent use, library preparation can be performed on a quarter of the scale described in the manufacturer's manual (i.e., by using 25 ng of template DNA) if the following conditions are used:Thermocycling for the fragmentation, end repair, and “A” addition steps is conducted under the following program: FX reaction mix is prepared following table without FX Enhancer. Amplification of library DNA is performed with the following condition.  ABC1StepTemperatureTime214℃1 min3232℃320 sec4365℃30 min544℃HoldFX reaction mix is prepared following table without FX Enhancer. Amplification of library DNA is performed with the following condition.  AB1ComponentAmount2FX Buffer, 10x1.25 μl3Purified DNA (in 10 mM Tris, pH 8.0)25 ng4Nuclease-free waterVariable5Total volume without FX Enhancer and FX Enzyme Mix10 μl
ABC1StepTemperatureTime214℃1 min3232℃320 sec4365℃30 min544℃Hold
AB1ComponentAmount2FX Buffer, 10x1.25 μl3Purified DNA (in 10 mM Tris, pH 8.0)25 ng4Nuclease-free waterVariable5Total volume without FX Enhancer and FX Enzyme Mix10 μl
?. [Preparation of the mitogenome libraries for next-generation sequencing]
Take an aliquot of each library and dilute it 1:3 with 10 mM Tris-HCl, pH 8.0 for quality check and quantification (described below). Store the rest of the libraries at −20℃ until use.
?. [Preparation of the mitogenome libraries for next-generation sequencing]
Check the quality and fragment size of each diluted library by using an Agilent BioAnalyzer (Agilent) and Agilent High Sensitivity DNA Kit (Agilent).Note: The quality (fragment size and presence of adaptor dimer) can also be checked in the next step 10.
?. [Preparation of the mitogenome libraries for next-generation sequencing]
Quantify the molar concentration of each library by conducting qPCR using an KAPA Library Quantification Kit (KAPA Biosystems)(please refer to the protocol (dx.doi.org/10.17504/protocols.io.bmgck3sw) for details)
?. [Preparation of the mitogenome libraries for next-generation sequencing]
Pool equal amounts of each library adjusted to 2 nM to obtain the final library.Note: It is recommended to check by qPCR whether the molar concentration of the pooled library is appropriate.
?. [Preparation of the mitogenome libraries for next-generation sequencing]
Sequence the pooled and denatured library (8 pM) containing 5% volume of PhiX (control library; Illumina) by using an Illumina MiSeq system and MiSeq Reagent Kit V3 (300 bp paired-end reads) (Illumina).
?. [Preparation of the mitogenome libraries for next-generation sequencing]
For each library, dissolve the amplified and purified library DNA in 10 mM Tris-HCl, pH 8.0.
?. [Preparation of the mitogenome DNA]
Amplify the Oncorhynchus masou masou whole-mitogenome fragment (16.6 kbp) with the following PCR mixture and PCR program:  AB1ComponentAmount25× PrimeSTAR GXL Buffer (TaKaRa Bio Inc.)3 μl3PrimeSTAR GXL DNA polymerase (TaKaRa Bio Inc.)0.3 μl42.5 mM dNTP mix1.2 μl510 μM primer MT-F0.3 μl610 μM primer MT-R0.3 μl7Genomic DNA20–100 ng8Nuclease-free waterVariable9Total volume15  μl
AB1ComponentAmount25× PrimeSTAR GXL Buffer (TaKaRa Bio Inc.)3 μl3PrimeSTAR GXL DNA polymerase (TaKaRa Bio Inc.)0.3 μl42.5 mM dNTP mix1.2 μl510 μM primer MT-F0.3 μl610 μM primer MT-R0.3 μl7Genomic DNA20–100 ng8Nuclease-free waterVariable9Total volume15  μl
?. [Preparation of the mitogenome DNA]
Electrophorese the total PCR product mixture through a 0.8% agarose–TAE gel.
?. [Preparation of the mitogenome DNA]
Measure the concentration of the purified mitogenome DNA by using a spectrophotometer. Also, check the quality of the purified mitogenome DNA by conducting agarose gel electrophoresis.
?. [Preparation of the mitogenome DNA]
Extract the mitogenome from the excised band by using the Wizard SV Gel and PCR Clean-up System (Promega), and elute with 20 μl 10 mM Tris-HCl, pH 8.0 warmed to 60℃.
?. [Sequence analysis]
Check the quality of the obtained next generation sequencing (NGS) data (i.e., FASTQ data) by using FastQC software.
?. [Sequence analysis]
Trim the FASTQ data by using Trimmomatic ver. 0.36 as follows: (a) apply a cleanup adapter and (b) filter out reads with low quality (Q score,
?. [Sequence analysis]
Map the reads to the reference mitogenome sequence (O. m. masou accession No: NC_008747) by using Burrow–Wheeler Aligner ver. 0.7.12.
?. [Sequence analysis]
Convert the obtained SAM (Sequence Alignment Map) files to BAM (Binary Alignment Map) files using SAMtools ver. 1.4.1.
?. [Sequence analysis]
Visualize the resulting reads with Integrative Genomics Viewer version 2.3.83 and identify SNP (single nucleotide polymorphism) sites with TASSEL ver. 5.0.
?. [Sequence analysis]
Replace the primer sites and those outside the region of interest (mitogenome positions: 13950–14022) with the predetermined ND5 sequence.
?. [Preparation of the mitogenome DNA]
Cut the band containing the mitogenome from the agarose gel.