I am trying to use Tax4Fun2 (T4F2) to perform functional prediction from 16S rRNA, ITS and COI gene sequencing. However, I can’t find a clear and working script. So far the ones I’ve found are giving errors. Can anybody help me?

library(Tax4Fun2)

# modify the following 4 lines as needed

pwd_ref_data = ‘Tax4Fun2_ReferenceData_v2’ # path to Tax4Fun2’s default database, need to be decompressed before use

pwd_user_data = ‘genome_folder’ # path to the folder that holds the reference genome/MAG files

name_user_data = ‘name_of_user_database’ # specify the name of the generated database, specify only the name, do not include path here!

gnm_ext = ‘fna’ # extension of the genome files

# Generate your own database

extractSSU(genome_folder = pwd_user_data, file_extension = gnm_ext, path_to_reference_data = pwd_ref_data)

assignFunction(genome_folder = pwd_user_data, file_extension = gnm_ext, path_to_reference_data = pwd_ref_data, num_of_threads = num_threads, fast = TRUE)

generateUserData(path_to_reference_data = pwd_ref_data, path_to_user_data = pwd_user_data, name_of_user_data = name_user_data, SSU_file_extension = “_16SrRNA.ffn”, KEGG_file_extension = “_funPro.txt”)

# modify the following 10 lines as needed

query_otu_seq = ‘demo_OTU.fasta’ # input OTU sequence file

query_otu_table = ‘demo_OTU_table.csv’ # input OTU table

pwd_op_folder = ‘Tax4Fun2_output_folder’ # output directory

pwd_ref_data = ‘Tax4Fun2_ReferenceData_v2’ # need to be the same as in step 1

pwd_user_data = ‘genome_folder’ # need to be the same as in step 1

name_user_data = ‘name_of_user_database’ # need to be the same as in step 1, specify only the name, do not include path here

norm_by_cn = TRUE # normalize_by_copy_number (TRUE or FALSE)

norm_path = TRUE # normalize_pathways (TRUE or FALSE)

iden = 0.97 # min_identity_to_reference, please modify as needed

num_of_threads = 6 # number of CPU cores to use , please modify as needed

# predict functions

runRefBlast(path_to_otus = query_otu_seq, path_to_reference_data = pwd_ref_data, path_to_temp_folder = pwd_op_folder, database_mode = “Ref99NR”, use_force = T, num_threads = num_of_threads, include_user_data = T, path_to_user_data = pwd_user_data, name_of_user_data = name_user_data)

makeFunctionalPrediction(path_to_otu_table = query_otu_table, path_to_reference_data = pwd_ref_data, path_to_temp_folder = pwd_op_folder, database_mode = “Ref99NR”, normalize_by_copy_number = norm_by_cn, min_identity_to_reference = iden, normalize_pathways = norm_path, include_user_data = T, path_to_user_data = pwd_user_data, name_of_user_data = name_user_data)