DNA was extracted from cecal content using the PowerSoil DNA extraction kit (MO BIO Laboratories, Carlsbad, CA, USA) following the manufacturer’s protocol. Between 400 and 500 ng of total DNA was used for library preparation for Illumina sequencing employing Illumina DNA Prep kit (Illumina, San Diego, CA, USA). All libraries were assessed using a TapeStation High Sensitivity DNA kit (Agilent Technologies, Santa Clara, CA, USA) and were quantified by Qubit (Invitrogen, Waltham, MA, USA).
Validated libraries were pooled in equimolar quantities and sequenced as a paired-end 150-cycle run on an Illumina NextSeq2000 (Illumina, San Diego, CA, USA). A total of 1548 million reads were generated, and raw reads were filtered for QV > 30 using an in-house phyton script. Filtered reads were aligned to unique clade-specific marker genes using MetaPhlAn 3 [16 (link)] to assess the taxonomic profile. Alignment was performed indicating the closest name of species to the sequence (the best hit). The relative proportions calculated from MetaPhlAn were used to calculate relative abundances, alpha diversity measure (chao1 index) and beta diversity measures (Aitchison distance).
Validated libraries were pooled in equimolar quantities and sequenced as a paired-end 150-cycle run on an Illumina NextSeq2000 (Illumina, San Diego, CA, USA). A total of 1548 million reads were generated, and raw reads were filtered for QV > 30 using an in-house phyton script. Filtered reads were aligned to unique clade-specific marker genes using MetaPhlAn 3 [16 (link)] to assess the taxonomic profile. Alignment was performed indicating the closest name of species to the sequence (the best hit). The relative proportions calculated from MetaPhlAn were used to calculate relative abundances, alpha diversity measure (chao1 index) and beta diversity measures (Aitchison distance).
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