According to some web sources¹²³⁴, some answers are:

- Yes, you need to combine your forward and reverse sequences before BLASTing them, because they are complementary and overlapping parts of the same 16S gene. You can use software such as ChromasPro, Snap Gene, Gene Edit, or Unipro Ugene to align and merge your sequences¹⁴. You can also use online tools such as this one² to merge your sequences in bulk.

- To determine which BLAST result to use as your identification of the organism, you need to look at the alignment score, the query coverage, and the percent identity of the matches. The alignment score reflects how well your query sequence matches the database sequence. The query coverage indicates how much of your query sequence is aligned to the database sequence. The percent identity shows how similar your query sequence is to the database sequence. Generally, you want to choose a result that has a high alignment score, a high query coverage, and a high percent identity. You also want to check the taxonomic information of the matches and see if they are consistent with each other and with your expectations.

(1) How can the forward and reverse sequence of an isolate be combined for .... https://www.researchgate.net/post/How_can_the_forward_and_reverse_sequence_of_an_isolate_be_combined_for_a_BLAST_analysis_to_be_performed.

(2) Nucleotide BLAST: Align two or more sequences using BLAST. https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq.

(3) Forward sequencing and reverse sequencing have different results. https://www.researchgate.net/post/Forward_sequencing_and_reverse_sequencing_have_different_results.

(4) How do I assemble F and R pairs of Sanger sequences?. https://help.geneious.com/hc/en-us/articles/360044626752-How-do-I-assemble-F-and-R-pairs-of-Sanger-sequences-.