Pe150 strategy

The genomic library of soybean cpDNA library was prepared for sequencing on Illumina platforms with PE150 strategy, following the manufacturer’s recommended protocol (Illumina, California, USA). After removal of DH10B genome sequences. The DNA library generated 3.25 Gb clean data and the average sequencing depth was 21,348.70x. The sequencing data was of high quality (Q 20 = 96.8%, Q 30 = 91.5%).
Bowtie2 v2.5.1 (https://bowtie-bio.sourceforge.net/bowtie2/index.shtml), Samtools v1.18 (http://www.htslib.org/), SPAdes v3.13.0 (https://cab.spbu.ru/software/spades/), A5-miseq v20160825 (https://sourceforge.net/projects/ngopt/files/), and Gapfiller v2.1.2 (https://sourceforge.net/projects/gapfiller/files/) assembly software were used separately to assemble the clean data. The primary assembly yielded a complete circular cpDNA of soybean. After annotation of regions by alignment against the soybean chloroplast Ref-genome (NC_007942) by the online software CPGAVAS2 (http://47.96.249.172:16019/analyzer/annotate), oligonucleotide primers were designed to amplify across problematic, remaining gap regions, and polymorphism site.

Isolation of gDNA was carried out using SDS method. Total DNA obtained was subjected to quality control by agarose gel electrophoresis and quantified by Qubit (Thermo Fisher Scientific, Waltham, MA, USA). The genome of L. lactis N8-8 was sequenced with MPS (massively parallel sequencing) Illumina technology (Illumina, San Diego, CA, USA). The DNA library was constructed: a paired-end library with an insert size of 350 bp. The 350-bp library was sequenced using an Illumina PE150 strategy. Library construction and sequencing were performed at the Beijing Novogene Bioinformatics Technology Co., Ltd. Quality control of paired-end reads was performed using in-house program. Then, data processing, reads mapping and SV (structural variation) analysis were conducted as we described previously [12 (link)].

We constructed libraries with a 350-bp insert fragment for G. thurberi and G. davidsonii according to the manufacturer’s instructions (Illumina). A HiSeq 2500 system was used to sequence the libraries, along with a PE150 strategy according to the manufacturer’s instructions (Illumina). The sequence adaptors were removed for the uncleaned Illumina reads, and the contaminated reads (viral, mitochondrial, bacterial sequences) were compared with the NCBI-NR database via BWA v0.7.13 [37 (link)] (using default instructions). Duplicate pairs were identified using FastUniq v1.12 [38 (link)]. In total, we produced 116.9 Gb and 118.2 Gb clean Illumina reads for G. raimondii and G. davidsonii, respectively. For CENH3 analysis, the raw sequencing data were downloaded from the Gene Expression Omnibus for G. hirsutum (accession number GSE119184) [39 ] and the European Molecular Biology Laboratory-European Bioinformatics Institute (accession number PRJEB14368) [40 ]. The data were aligned to the reference genome with Botiwe2, and the enrichment was calculated by dividing the CENH3 read counts by the input read counts according to previously used methods [28 (link)].

Total microbial genomic DNA samples were extracted using the OMEGA Mag-Bind Soil DNA Kit (M5635-02) (Omega Bio-Tek, Norcross, GA, USA), following the manufacturer’s instructions, and stored at -20 °C prior to further assessment. The quantity and quality of extracted DNAs were measured using a Qubit™ 4 Fluorometer, with WiFi: Q33238 (Qubit™ Assay Tubes: Q32856; Qubit™ 1X dsDNA HS Assay Kit: Q33231) (Invitrogen, USA) and agarose gel electrophoresis, respectively. The extracted microbial DNA was processed to construct metagenome shotgun sequencing libraries with insert sizes of 400 bp by using Illumina TruSeq Nano DNA LT Library Preparation Kit. Each library was sequenced by Illumina NovaSeq platform (Illumina, USA) with PE150 strategy at Personal Biotechnology Co., Ltd. (Shanghai, China).

Total microbial genomic DNA samples were isolated using the E.Z.N.A Soil DNA kit (Omega Bio-tek, Norcross, GA, USA) (D5625-01), according to the manufacturer’s protocol. The isolated DNA was stored at -20 ° C. The prepared sample buffer was also subjected to laboratory-controlled extraction to identify any potential contaminants. The quantity and quality of the extracted DNAs were measured using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively. The extracted microbial DNA was processed using the Illumina TruSeq Nano DNA LT Library Preparation Kit to construct metagenome shotgun sequencing libraries with insert sizes of 400 bp. Each library was sequenced using the Illumina NovaSeq platform (Illumina, USA) with PE150 strategy at Personal Biotechnology Co. Ltd. (Shanghai, China).

The total DNA was extracted from the insects' whole body tissue using the Tissue DNA Kit (Omega Georgia, Connecticut, USA) according to the manufacturer's instructions. The DNA content was quantified using a NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Scientific, Massachusetts, USA). A measure of 0.2 μg DNA was used as the input material for DNA library preparation. The sequencing library was prepared using the NEBNext UltraTM DNA Library Prep Kit (New England Biolabs, New York, USA) for Illumina according to the manufacturer's recommendations, and index codes were added. DNA fragments were then end-polished, A-tailed and ligated with the full-length adapter for Illumina sequencing, followed by further polymerase chain reaction (PCR) amplification. After PCR, the products were purified using the AMPure XP system (Beverly, Los Angeles, USA). The quality of the libraries was then assessed using the Agilent 5400 system (Agilent, Palo Alto, USA) and quantified by quantitative PCR (1.5 nm). The qualified libraries were pooled and sequenced on Illumina platforms with the PE150 strategy at Novogene Bioinformatics Technology Co., Ltd. (Beijing, China) according to the effective library concentration and the required amount of data.