RNA-Seq of Poultry
Total genome DNA from samples was extracted using CTAB method. DNA concentrationand purity was monitored on 1% agarose gels. According to the concentration, DNA was dilutedto 1ng/L using sterile water. 1.2 Amplicon Generation16S rRNA/18SrRNA/ITS genes of distinct regions(16S V4/16S V3/16S V3-V4/16S V4-V5, 18S V4/18S V9, ITS1/ITS2, Arc V4) were amplified used specific primer(e.g. 16S V4: 515F- 806R, 18S V4: 528F-706R, 18S V9: 1380F- 1510R, et. al ) with the barcode. All PCR reactionswere carried out with 15 L of Phusion High -Fidelity PCR Master Mix (New England Biolabs);2 M of forward and reverse primers, and about 10 ng template DNA. Thermal cycling consistedof initial denaturation at 98C for 1 min, followed by 30 cycles of denaturation at 98C for 10 s, annealing at 50C for 30 s, and elongation at 72C for 30 s. Finally 72C for 5 min. 1.3 PCR Products quantification and qualificationMix same volume of 1X loading buffer (contained SYB green) with PCR products andoperate electrophoresis on 2% agarose gel for detection. PCR products was mixed inequidensity ratios. Then, mixture PCR products was purified with Qiagen Gel ExtractionKit(Qiagen, Germany). 1.4 Library preparation and sequencingSequencing libraries were generated usingTruSeq DNA PCR-Free Sample Preparation Kit(Illumina, USA) following manufacturer's recommendations and index codes were added. Thelibrary quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and AgilentBioanalyzer 2100 system. At last, the library was sequenced on an Illumina NovaSeq platformand 250 bp paired-end reads were generated. 2. Bioinformatics Analysis PipelinePaired-end reads was assigned to samples based on their unique barcode and truncated bycutting off the barcode and primer sequence. 2.1.2 Sequence assemblyPaired-end reads were merged using FLASH (V1.2.11, http://ccb.jhu.edu/software/FLASH/)(Magoc T et al.,2011), a very fast and accurate analysis tool, which was designed to mergepaired-end reads when at least some of the reads overlap the read generated from the opposite endof the same DNA fragment, and the splicing sequences were called raw tags. 2.1.3 Data FiltrationQuality filtering on the raw tags were performed using the fastp (Version 0.23.1) software toobtain high-quality Clean Tags( Bokulich NA et al.,2012). 2.1.4 Chimera removalThe tags were compared with the reference database(Silva database (16S/18S), https://www.arb-silva.de/; Unite Database(ITS), https://unite.ut.ee/) using UCHIME Algorithm(http://www.drive5.com/usearch/manual/uchime_algo.html)to detect chimera sequences, and thenthe chimera sequences were removed (Edgar RC et al.,2011). Then the Effective Tags finallyobtained.