Computational protocol: Specific Microbiome Changes in a Mouse Model of Parenteral Nutrition Associated Liver Injury and Intestinal Inflammation

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Protocol publication

[…] Fecal samples were obtained from the descending colon on the day of sacrifice (after 7 days of PN or chow diet) using sterile technique and snap frozen and stored at −70°C. Total fecal DNA was extracted as previously described . DNAs were amplified in triplicate using barcoded primers (27F/338R) that target the V1–2 region of the small subunit ribosomal RNA gene . A negative PCR control was run for each individual barcode. Amplicon concentration was normalized using the SequalPrep plate (Invitrogen) prior to mixing in equal amounts . Sequencing was performed using the 454 titanium system (Roche) using manufacturer’s protocols. Sequence data was assigned to the appropriate sample and initial quality checks were performed using BARTAB . The Omegaven and no lipid control mice were analyzed with the identical region of the rRNA gene, but with adapters compatible with the Illumina MiSeq platform. PCR was performed as described above. Pooled amplicons were quantified using the Qubit Fluorometer 2.0 (Invitrogen, Carlsbad, CA). The pool was diluted to 2 nM and denatured with 0.2 N NaOH at room temperature. The denatured DNA was diluted to 15 pM and spiked with 25% of the Illumina PhiX control DNA prior to loading the sequencer. Illumina paired-end sequencing was performed on the MiSeq platform with version 2.0 of the MiSeq Control Software, using a 500-cycle version 2 reagent kit. MiSeq paired-end sequences were sorted by sample via barcodes in the paired reads with a python script. The sorted paired reads were assembled using phrap . All sequence ends (454 or MiSeq) were trimmed over a moving window of 5 nucleotides until average quality met or exceeded 20. Trimmed sequences with more than 1 ambiguity or shorter than 200 nt were discarded. Potential chimeras identified with Uchime (usearch6.0.203_i86linux32) using the Schloss Silva reference sequences were removed from subsequent analyses. Assembled sequences were aligned and classified with SINA (1.2.11) using the 244,077 bacterial sequences in Silva 111NR as reference configured to yield the Silva taxonomy. Sequences with identical taxonomic assignments were grouped to produce Operational taxonomic units (OTUs). The software package Explicet (v2.8; www.explicet.org ) was used for display, analysis, and figure generation of results. Sequence data is available from NCBI Sequence Read Archive under accession SRP041319. [...] Each taxon was evaluated using the Kruskal-Wallis test across all groups and used FDR correction for multiple comparisons. Random forests (RF) is a popular method employed in high dimensional datasets to identify complex data . We used RF here to identify important genera for classification in a multivariate fashion. A random forest (RF) of 10,000 trees was implemented using the RandomForest R-package, all other default parameters were used. Principal component analysis was performed on the relative abundance data from each mouse. A small constant (1/total number of sequences) was added to the counts prior to the application of the centered log ratio transformation recommended for compositional data , .Recently studies in other mouse models of liver injury have detected high levels of sequences identified only as “Bacteria” without further details, suggesting limitations with standard approaches of assessing the fecal microbiome in other mouse disease models , . In contrast, in the present study we utilized expanded reference sequences to identify the bacteria present after standard classification approaches failed to provide identification for a significant number of sequences. This highlights the importance of the tools used to analyze microbiome data, and the need for alternative approaches when standard approaches are not successful . Our expanded reference set identified a lineage of Bacteroidetes (S24-7 ) that was not represented in the reference taxa used for chimera detection . Others have noted the importance of reference taxa selection . […]

Pipeline specifications

Software tools XSTK, UCHIME, USEARCH, Explicet, randomforest
Databases SRA
Applications Miscellaneous, Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Mus musculus, Homo sapiens
Diseases Malnutrition, Chemical and Drug Induced Liver Injury
Chemicals Sterols, Stigmasterol