Table 1 Considerations for new users developing a helminth metabarcoding protocol
Step | Method | Prosa | Consa |
|---|---|---|---|
1. Collect samples | Fecal material | Non-invasive | Risk of environmental contamination |
Low effort to collect | Parasite species that are actively reproducing may make up larger portion of sequence reads | ||
Can obtain a large sample size | DNA may be degraded and/or PCR inhibitors may be present in fecal matter | ||
Gastrointestinal tract tissue | Can detect parasites that are not actively reproducing | Invasive | |
Environmental contamination is limited | Obtaining a large sample size may be difficult, especially for imperiled species | ||
Cloacal swab | Non-invasive | Only available for certain host species | |
Low effort to collect | Swab may miss parasite species located higher up in orifice | ||
2. Extract DNA | Traditional technique (i.e. isopropanol precipitation, phenol–chloroform) | May already have reagents in laboratory, low cost | May need to troubleshoot to validate protocol |
High DNA yield | Lower detection rate of species present in sample | ||
Commercial kit | DNA kits made specifically for fecal matter to decrease PCR inhibitors | Higher cost | |
High detection rate of species present in sample | Variable DNA yield depending on sample type and kit used | ||
Isolating parasites from sample before extraction | Limits PCR inhibitors in fecal matter | May lose some adult parasites if utilizing a sieving method focused on collected eggs, avoidable if using co-proculture and extracting whole sample | |
Time-consuming | |||
Lysis procedures (i.e. bead beating) | Increase DNA yield of species with hard eggshells | Time-consuming, additional costs | |
3. Amplified marker region | Nuclear ITS | Good for species identification due to highly variable sequences between species | ITS region may not as suitable for inferring phylogenetic relationships due to nucleotide substitution saturation |
Nuclear rRNA (28S, 18S) | Highly conserved, good for determining phylogenetic relationships | Lower resolution of species-level identification | |
Easier primer design due to more universal primers and reference sequences available | Only a small proportion of sequence reads are specific to nematodes for 18S | ||
Mitochondrial COX1 | Universal primers available | High sequence variation in COX1Â gene in helminths compared to other groups leads to low PCR amplification success and limited taxa identification | |
Mitochondrial 16S | Good phylogenetic and species resolution | Used less frequently in studies causing fewer reference sequences available | |
Mitochondrial 12S | Good phylogenetic and species resolution, may recover more platyhelminth and nematode species than 16S | Used less frequently in studies causing fewer reference sequences available | |
4. Sequencing platforms | Illumina (Illumina Inc., San Diego, CA, USA) | Low error rates | Maximum read length is appprox. 500 bp (for paired end sequencing) |
Produces short reads, which can provide high read numbers per sample | Short reads may have lower accuracy in assigning taxonomy | ||
Available at most genomic core facilities, well-established procedures | Less power in determining phylogenetic relationships | ||
Pacific Biosciences of California, Inc. (PacBio; Menlo Park, CA, USA) | Produces long reads, up to 10,000Â bp long | Primer pairs need to be developed to cover the longer fragments, lower read depth | |
Long reads may be necessary if using long marker regions or a combination of marker regions | Less available at genomic core facilities than Illumina platforms | ||
Long reads are better for phylogenetic inferences | Higher error rate than the Illumina method | ||
5. Bioinformatic analysis and database | NCBI GenBank | Database is largest, contains sequences from all phyla | Not all sequences are annotated |
Compatible with any genetic marker | Mislabeled sequences may be present, database is less curated than more custom databases | ||
Nemabiome ITS2 database | Clear protocols developed | Only contains sequences from phylum Nematoda | |
Database is updated frequently | Only compatible with ITS2 genetic marker | ||
SILVA rRNA database | Quality checked and regular updates | Only compatible with rRNA markers (18S, 16S, 28S, 23S) | |
Protist Ribosomal Reference database (PR2) | Annotated, allows for interpretation of the structure and function of specific genes | Only compatible with 18S rRNA and limited 16S genetic markers |