Tapestation high sensitivity dna kit

Illumina-compatible indexed libraries were prepared from 500 ng of Biorupter Ultrasonicator (Diagenode)-sheared gDNA using the KAPA Hyper Library Preparation Kit (Kapa Biosystems, Wilmington, MA, USA). Library quality was assessed using the TapeStation High Sensitivity DNA Kit (Agilent Technologies). Exome capture of the library pool was performed using the NimbleGen SeqCap EZ Exome kit V3.0 (Roche-Nimblegen, Madison, WI, USA). Sequencing was then performed in one lane of the HiSeq3000 Sequencer (Illumina Inc., San Diego, USA) using the 76 nt paired end format.

DNA was extracted from faeces 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. 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 [80 (link)] to assess the taxonomic profile. The alignment was done 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).

The shotgun metagenomic sequencing was performed in 8 animals per group. DNA was extracted from cecum 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 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. 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 [55 (link)] to assess the taxonomic profile. The 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 measure (Aitchison distance).