Ultrasonic crusher

The hyper-variable V3–V4 region of the 16S rDNA gene was amplified by PCR using primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′). The PCR products were purified and quantified using a QuantiFluorTM fluorometer (Promega, Madison, WI, USA). Negative controls for PCR amplification were reactions without DNA. The PCR products were purified and quantified using a fluorometer (QuantiFluor; Promega, Madison, WI, USA) and then sequenced on a HiSeq 2500 platform using the PE250 model (Novogene, Beijing, China). The qualified DNA samples were randomly sheared to a length of approximately 350 bp using a Covaris ultrasonic crusher (Woburn, MA, USA). The entire library was prepared using the following steps: end repair, adding a 3′ poly-A tail, ligating adapters, purification, and PCR amplification. Library quality was assessed on a Qubit 2.0 Fluorometer (Thermo Scientific, Waltham, MA, USA) and Agilent Bioanalyzer 2100 system (Agilent Technologies, Palo Alto, CA, USA). The library was sequenced on the Illumina HiSeq platform (San Diego, CA, USA). The sequencing produced an average of 540 million reads (approximately 12 GB) per sample.

The QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) was used to extract microbial DNA from the sable stool samples. Before library construction, the DNA was evaluated for quality control and quantified. Agarose gel electrophoresis (AGE) was used to analyse the purity and integrity of the DNA, and Qubit 2.0 (Invitrogen, USA) was used to precisely quantify DNA concentration. During library construction, qualified DNA samples were randomly broken into fragments approximately 350 bp in length with an ultrasonic crusher (Covaris, UK). Then, the fragments were end-repaired, A-tailed, and ligated to adapters. After library preparation, Qubit 2.0 (Invitrogen, USA) was used for initial quantification, and the library was diluted to 2 ng/µl. Subsequently, an Agilent 2100 Bioanalyzer (Agilent, USA) was used to determine whether the insert sizes of the library corresponded to expectations. To ensure library quality, real-time q-PCR was used to accurately quantify the effective concentration (> 3 nM) of the library. After the library passed the inspection, sequencing was implemented on an Illumina HiSeq X Ten platform (Illumina, USA). The raw reads are available at the NCBI Sequence Read Archive (BioProject ID PRJNA630144, SRA SRP265006).

Total genomic DNA was extracted from silica‐dried leaves by the DNA quick extraction system (DP321) according to the manufacturer's protocol (Tiangen Biochemical Technology Co., Ltd.). Total DNA was then randomly fragmented with the Covaris ultrasonic crusher. A series of steps were performed to complete library construction, such as end repair and phosphorylation, a‐tail addition, sequencing connector addition, purification, and PCR amplification. Finally, the qualified libraries were pooled into flowcells. NovaSeq 6000 (Illumina Inc.) was used for sequencing after cBOTs clustering, with paired‐end methods (150 bp). We used fastp v.0.23.1 (Chen et al., 2018 (link)) to filter raw sequence reads when: (i) the N content in any read was more than 10% of the base; (ii) the number of low quality (Q ≤ 5) bases in any read exceeded 50%; and (iii) any read contained the adapter content; if so, the paired reads were removed (Yan et al., 2013 (link)).

DNA was sheared to 300 bp by the Covaris ultrasonic crusher. To prepare the sequencing library, the fragments were dealt with end repair, a tailing, and were ligated to the Illumina compatible adapters. The libraries of DNA sequencing were sequenced by Illumina Hiseq platform at Allwegene Company (Beijing). After the operation, image analysis, base calling, and error estimation were performed utilizing Illumina Analysis Pipeline Version 2.6.

Use 1% agarose gel electrophoresis (AGE) to analyze the purity and integrity of DNA, and use Qubit^® dsDNA Assay Kit in Qubit^® 2.0 Fluorometer (Life Technologies, CA, USA) to check DNA for quantification. Take an appropriate amount of sample into a centrifuge tube, and dilute the sample with sterile water until the OD value is between 1.8-2.0. Take 1μg genome DNA of the sample and use NEBNext^® Ultra, DNA Library Prep Kit for Illumina (NEB, USA) to construct the library. The genomic DNA was randomly sheared into fragments with a length of about 350 bp using Covaris ultrasonic crusher. The obtained fragments were end-repaired, A-tailed, and further ligated with a sequence adapter. The fragments with adapters were PCR amplified, size selected, and purified to construct the library. The constructed library was checked with Qubit2.0 for quantification, diluted to 2ng/ul, and then the insert size of the library was detected with Agilent 2100. After the insert size meets the expectation, the Q-PCR method is used to accurately quantify effective library concentration (effective library concentration is>3nM) to ensure the quality of the library. Quantified libraries will be pooled and sequenced on Illumina PE150 platforms, according to effective library concentration and data amount required.

PAMs were seeded as described and were infected with ASFV-WT or ASFV-Δ360-9L. DNA was isolated as described above from cells infected with the either of the viruses. Then, whole genome sequencing of the ASFV was performed (42 (link)). The isolated DNA was broken into segments using the Covaris ultrasonic crusher. In addition, a DNA library was prepared via terminal repair, adding poly-A tail, adding sequencing connector, purification, and PCR amplification. After the completion of library construction, the library was initially quantified using Qubit v.2.0 and diluted to 2 ng/μl; then, the insert fragment size of the library was detected via Agilent 2100. The effective concentration of the library was accurately quantified using the qPCR to ensure library quality. The DNA library was sequenced using Illumina HiSeq. The sequencing data were assembled using the SPAdes (v.3.13.0) software. The complete and accurate deletion MGF360-9L in ASFV-Δ360-9L was confirmed using whole-genome sequencing. There were no undesirable genetic changes in the virus (Data Set S1 in the supplemental material).