Ambiguous sequences at different cut off levels - (Item E)

For the evaluation of ambiguous sequences in microbial reference genomes we assessed different cut of levels for the generation of a curated microbial genome database. For each sequence fragment we determined the ratio between the number of negative BLAST hits (query genus ≠ subject genus) and all BLAST hits.<br><br>For a detailed description of this analysis, see the Supplemental Materials section of the publication.<br><br><b>Reference</b> <p><b>Kirstahler P, Bjerrum SS, Friis-Møller A, </b><b>la Cour M, Aarestrup FM, Westh H., and Pamp SJ. (2018) </b>Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid.<b> </b><b>Scientific Reports</b>, doi:10.1038/s41598-018-22416-4.</p

Ambiguous sequences in public microbial genomes - Sequence List - (Item D)

Scaffolds and contigs predicted to be ambiguous in the 5754 public microbial genomes examined in Kirstahler <i>et al</i>. (2017).<br><br><p><b>Reference</b></p> <p><b>Kirstahler P, Bjerrum SS, Friis-Møller A, </b><b>la Cour M, Aarestrup FM, Westh H., and Pamp SJ. (2018) </b>Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid.<b> </b><b>Scientific Reports</b>, doi:10.1038/s41598-018-22416-4.</p

Read counts for most abundant microbial agent in patient samples - (Item G)

Vitreous was analyzed using metagenomics using two DNA isolation methods (QIAamp DNA Mini Kit, QIA; QIAamp UCP Pathogen Mini kit, UCP) and the taxonomic affiliation of reads was determined using Kraken, Bracken, and BLASTn analysis. <br>The read counts for the most abundant microbial agent in endophthalmitis patients are listed. <br><br><p><b>Reference</b></p> <p><b>Kirstahler P, Bjerrum SS, Friis-Møller A, </b><b>la Cour M, Aarestrup FM, Westh H., and Pamp SJ. (2018) </b>Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid.<b> </b><b>Scientific Reports</b>, doi:10.1038/s41598-018-22416-4.</p

Comparison between QIAamp DNA Mini Kit [QIA], QIAamp UCP Pathogen Mini kit [UCP] - (Item O)

Bayesian estimation analysis of reads counts obtained from samples extracted with both, QIAamp DNA Mini Kit [QIA], QIAamp UCP Pathogen Mini kit [UCP]. <br><br> The analysis was carried out according to BEST by John K. Kruschke (Journal of Experimental Psychology: General, 2013, v.142(2), pp.573-603. doi: 10.1037/a0029146).<br><br><p><b>Reference</b></p> <p><b>Kirstahler P, Bjerrum SS, Friis-Møller A, </b><b>la Cour M, Aarestrup FM, Westh H., and Pamp SJ. (2018) </b>Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid.<b> </b> <b>Scientific Reports</b>, doi:10.1038/s41598-018-22416-4.</p><br><br

Bacteriophages/viruses in endophthalmitis and control samples - (Item L)

Bacteriophages (DNA viruses) detected in vitreous from endophthalmitis-positive and -negative patients, basal salt solution samples, and DNA extraction controls.<b><br><br>Reference</b> <p><b>Kirstahler P, Bjerrum SS, Friis-Møller A, </b><b>la Cour M, Aarestrup FM, Westh H., and Pamp SJ. (2018) </b>Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid.<b> </b><b>Scientific Reports</b>, doi:10.1038/s41598-018-22416-4.</p

Comparison of bioinformatics workflows (Item P)

Comparison of the performance of the Kraken+Bracken workflow described in Kirstahler <i>et al</i>. 2018, with 12 metagenomic classifiers using a synthetic mock community, as described in Peabody <i>et al.</i>, 2015. <br>Examination of the MetaSimHC250 dataset containing simulated reads from 11 microorganisms with a read length of 250 bp (Peabody <i>et al</i>., 2015). The values obtained using our Kraken+Bracken workflow (see Fig. 2 in Kirstahler <i>et al</i>., 2018) are compared with the values obtained by Peabody <i>et al</i>., 2015.<br>A) The proportion of assigned reads after classification. Kraken+Bracken performance is similar to Kraken.<br>B) Calculated sensitivity for tested methods on each individual species in the synthetic mock community. Our workflow shows comparable performance to the other Kraken results.<br>C) Calculated precision for tested methods on each individual species in the synthetic mock community. Kraken-based methods show high similarities.<br>D) Bracken results for MetaSimHC250 dataset with calculated sensitivity and precision for the classification of the Kraken+Bracken workflow.<br><br><p><b>Reference</b></p> <p><b>Kirstahler P, Bjerrum SS, Friis-Møller A, </b><b>la Cour M, Aarestrup FM, Westh H., and Pamp SJ. (2018) </b>Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid.<b> </b> <b>Scientific Reports</b>, doi:10.1038/s41598-018-22416-4. </p

Targeted PCR on vitreous DNA - (Item H)

In another independent assessment regarding the most abundant organism in the vitreous samples we employed targeted PCR assays to detect the two most frequently identified bacteria, <i>Staphylococcus epidermidis</i> and <i>Enterococcus faecalis</i>. For the detection of <i>S. epidermidis</i> the species-specific primers SE705-1 (5’-ATC AAA AAG TTG GCG AAC CTT TTC A-3’) and SE705-2 (5’-CAA AAG AGC GTG GAG AAA AGT ATC A-3’) were used (Martineau <i>et al.</i> 1996). For the detection of <i>E. faecalis</i> the species-specific primers <i>ddl</i>-E1 (5’-ATC AAG TAC AGT TAG TCT-3’) and <i>ddl</i>-E2 (5’-ACG ATT CAA AGC TAA CTG-3’) were used (Dutka-Malen <i>et al</i>., 1995).<br><br><p><b>Reference</b></p> <p><b>Kirstahler P, Bjerrum SS, Friis-Møller A, </b><b>la Cour M, Aarestrup FM, Westh H., and Pamp SJ. (2018) </b>Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid.<b> </b><b>Scientific Reports</b>, doi:10.1038/s41598-018-22416-4.</p

Classification of microbial genomes against human reference genome - (Item A)

Public microbial reference genomes were classified against the human reference genome using Kraken. Most contigs and scaffolds with a high kraken label have a size below 10.000 bases.<br><br><p><b>Reference</b></p> <p><b>Kirstahler P, Bjerrum SS, Friis-Møller A, </b><b>la Cour M, Aarestrup FM, Westh H., and Pamp SJ. (2018) </b>Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid.<b> </b><b>Scientific Reports</b>, doi:10.1038/s41598-018-22416-4.</p

Virus genomes in extended database - (Item K)

Viral genome sequences (7214) as part of the microbial genomes database used in the study:<br><b><br>Kirstahler P, Bjerrum SS, Friis-Møller A, </b><b>la Cour M, Aarestrup FM, Westh H., and Pamp SJ. (2018) </b>Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid. <b>Scientific Reports</b>, doi:10.1038/s41598-018-22416-4

Ambiguous sequences in public microbial genomes - Overview - (Item C)

Microbial reference sequences (archaea, bacteria, fungi, and protozoa from NCBI RefSeq database (20.12.2016)) were split into separate contigs at stretches of 10 Ns or more. Contigs and scaffolds below 10 kb (original and new as the result of split) were aligened against the non-redundant nucleotide collection (nt) database from NCBI using BLASTn with a minimal e-value of 1e-6. Hits with less than 70% query coverage where discarded from further analysis. Hits where assigned their corresponding genus level (e.g. Bradyrizobium for Bradyrizobium lablabi), and if a hit was already assigned to genus or higher level the level was not changed. The best hit for a query sequence was determined according to the bitscore. Query sequences with only hits within their respective genus where regarded as non-ambiguous. The remaining sequences were regarded as ambiguous and removed before building a Kraken and a microbial BLAST microbial reference database.<br><br><p><b>Reference</b></p> <p><b>Kirstahler P, Bjerrum SS, Friis-Møller A, </b><b>la Cour M, Aarestrup FM, Westh H., and Pamp SJ. (2018) </b>Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid.<b> </b><b>Scientific Reports</b>, doi:10.1038/s41598-018-22416-4.</p