*2.9. Prediction of Target Genes, Proteome Re-Annotation, Gene Ontology (GO), and KEGG Enrichment Analyses*

TargetSpy [44], MIRANDA [45], and PITA [46] were used in miRNAconsTarget from sRNAtoolbox to predict genes regulated by tick salivary gland miRNAs up- or downregulated in the in silico analysis [47]. Targets common to all three programs were further considered. In silico target prediction provided a high number of false positives, but cross-species comparisons and combinatorial effects reduced this number [48]. Target gene networks and KEGG pathways significantly enriched for target genes were extracted using the STRING [49] output. PANNZER2 [50] was used to functionally re-annotate the proteome of up- or downregulated genes (targets of detected miRNAs), and WEGO [51] was used to analyze and plot gene ontology (GO) annotations.

## **3. Results and Discussion**

## *3.1. Profile Characteristics of Small RNA Libraries*

There were 216,292,174 raw small RNA reads from *B. burgdorferi*-infected salivary glands and 212,542,697 from uninfected salivary glands. After adapter trimming and removal of short reads (≤18 nucleotides (nt)), 133,465,828 small RNA reads were available from *B. burgdorferi*-infected samples and 163,852,135 from uninfected samples for downstream analysis. The read length distribution shows the types of small RNAs present in *B. burgdorferi*-infected and uninfected salivary gland samples. Two main peaks at 22 nt (miRNAs/siRNAs) at 29 nt (piRNAs) were distinguishable in *B. burgdorferi*-infected and uninfected samples (Figure 1A). There were ~5 <sup>×</sup> <sup>10</sup><sup>7</sup> and ~2.5 <sup>×</sup> <sup>10</sup><sup>7</sup> 22 nt miRNA se-

quences in uninfected and infected salivary gland samples, respectively. The read length distribution in uninfected samples was comparable to infected samples. uninfected and infected salivary gland samples, respectively. The read length distribution in uninfected samples was comparable to infected samples.

There were 216,292,174 raw small RNA reads from *B. burgdorferi*-infected salivary glands and 212,542,697 from uninfected salivary glands. After adapter trimming and removal of short reads (≤18 nucleotides (nt)), 133,465,828 small RNA reads were available from *B. burgdorferi*-infected samples and 163,852,135 from uninfected samples for downstream analysis. The read length distribution shows the types of small RNAs present in *B. burgdorferi*-infected and uninfected salivary gland samples. Two main peaks at 22 nt (miR-NAs/siRNAs) at 29 nt (piRNAs) were distinguishable in *B. burgdorferi*-infected and uninfected samples (Figure 1A). There were ~5 × 107 and ~2.5 × 107 22 nt miRNA sequences in

*Int. J. Mol. Sci.* **2022**, *23*, 5565 5 of 16

*3.1. Profile Characteristics of Small RNA Libraries* 

**3. Results and Discussion** 

**Figure 1.** (**A**) Small RNA sequence length distribution in uninfected (clean) and *Borrelia-burgdorferi*infected *Ixodes scapularis* salivary glands. MicroRNAs are twenty-two (22) nucleotides in length. Clean, uninfected salivary glands; infected, *Borrelia-burgdorferi*-infected salivary glands. (**B**) Summary of the abundance of combined small RNA reads from *Ixodes scapularis* salivary glands matching various small RNA categories. Reads from uninfected and *Borrelia-burgdorferi*-infected samples are combined together for abundance calculation. **Figure 1.** (**A**) Small RNA sequence length distribution in uninfected (clean) and *Borrelia-burgdorferi*infected *Ixodes scapularis* salivary glands. MicroRNAs are twenty-two (22) nucleotides in length. Clean, uninfected salivary glands; infected, *Borrelia-burgdorferi*-infected salivary glands. (**B**) Summary of the abundance of combined small RNA reads from *Ixodes scapularis* salivary glands matching various small RNA categories. Reads from uninfected and *Borrelia-burgdorferi*-infected samples are combined together for abundance calculation.

#### *3.2. Other Small RNA Categories 3.2. Other Small RNA Categories*

A summary of reads matching various small RNA categories from *B. burgdorferi*-infected and uninfected salivary glands is shown in Figure 1B. Other small RNAs include signal recognition particle rRNAs, ncRNAs, protein-coding RNAs, snoRNAs, snRNA SRP RNAs, and not-annotated small RNAs. Of the total small RNA reads from *Ixodes scapularis* salivary glands, 12.3% were miRNAs, 2.2% rRNAs, 0.4% ncRNAs, 0.8% protein coding, 0.07% snoRNAs, 0.05% snRNAs, 0.04% SRP RNAs, and 84.3% reads were not annotated. A summary of reads matching various small RNA categories from *B. burgdorferi*infected and uninfected salivary glands is shown in Figure 1B. Other small RNAs include signal recognition particle rRNAs, ncRNAs, protein-coding RNAs, snoRNAs, snRNA SRP RNAs, and not-annotated small RNAs. Of the total small RNA reads from *Ixodes scapularis* salivary glands, 12.3% were miRNAs, 2.2% rRNAs, 0.4% ncRNAs, 0.8% protein coding, 0.07% snoRNAs, 0.05% snRNAs, 0.04% SRP RNAs, and 84.3% reads were not annotated.
