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Lugli et al. Microbiome Res Rep 2023;2:15  https://dx.doi.org/10.20517/mrr.2022.21  Page 11 of 16



 Table 3. MEGAnnotator2 report of 10 sequentially processed microbial genomes using short- and long-read technologies

 High
 Sequencing                          Average
 High   quality  16S rRNA                       Number              Number  Number  Number
 Sequencing   output   ANI   Genome   Genome   Average  coverage   Genome
 SRA  quality   reads  gene                     of                  of       of rRNA  of tRNA
 output:  (long   screening: completeness: contamination: coverage: (long   length:
 reads:  (long   identity:                      contigs:            genes:   genes:    genes:
 reads):                             reads):
 reads):
 ERR5950940 1000000  973651        Shigella boydii  Shigella   99.97%  0.75%  75  39  55  6,606,912 6,860  22  97
 73857  42648  99.9%  boydii 98.2%

 ERR5951433  559060  514029  198629  60015  Klebsiella   Klebsiella   100%  1.04%  45  86  4  5,595,850 5,217  25  88
 pneumoniae   pneumoniae
 99.9%  99.0%
 SRR12326962 369346  357819  92666  62330  Salmonella   Salmonella   100%  0.78%  32  97  5  5,310,747 5,134  22  92
 enterica 100% enterica
 98.8%
 SRR12811523  1000000  991228  196879  106628 Enterobacter   Klebsiella   100%  1.11%  49  99  8  5,567,807 5,281  25  90
 aerogenes   aerogenes
 99.9%  95.8%
 SRR12811532  1000000  995953  102851  77655  Citrobacter   Citrobacter   99.94%  1.86%  48  99  16  5,693,281 5,598  25  86
 freundii 100% portucalensis
 94.7%

 SRR12811547  1000000  996250  222219  79032  Enterobacter   Enterobacter  98.92%  1.06%  50  98  19  5,496,621 5,248  25  87
 cloacae 100% cloacae
 99.7%
 SRR13177364 1000000  962158304 158304  61251  Kocuria   n.d.  98.68%  0%  163  120  2  2,848,142 2,432  9  48
 varians 100%

 SRR13249533 850449  845436  109409  43592  Bacillus   Heyndrickxia  98.30%  6.27%  76  68  2  5,364,016 5,378  36  145
 oleronius   oleronia
 99.8%  100%
 SRR14087463 1000000  991529  90337  62461  Pseudomonas  Pseudomonas  99.60%  0.17%  41  66  14  6,729,107 6,213  12  68
 aeruginosa   aeruginosa
 100%  94.1%
 SRR21755520 1000000  944753  137078  25119  Agrobacterium  n.d.  97.26%  3.31%  46  52  4  5,773,279 5,401  15  60
 arsenijevicii
 98.9%




 microbial species above used to validate the new pipeline version [Table 1]. An updated RefSeq database was downloaded from NCBI and formatted using

 Rapsearch2 as reported in the MEGAnnotator manual to compare the pipeline efficiency. Furthermore, the last version of the MIRA assembler has been
 installed on the same machine used for MEGAnnotator2 benchmarking.
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