Question 1:

If my mitochondrial genome has multiple independent circular contigs, or a complex network structure composed of multiple contigs, how can I use IPMGA to annotate it?

Answer: Please note that plant mitochondrial genomes exhibit complex dynamic changes, and you may use some software to obtain genomic graph with complex network structures. Some plant mitochondrial genomes also exhibit multiple independent circular contigs, such as Gastrodia elata (19 circular contigs: MF070084.1-MF070102.1). For all contigs representing the complete mitochondrial genome can be integrated into a single FASTA file and submitted to IPMGA. The following is an example of a FASTA-formatted file containing multiple sequences:

>contig1
GCATTCTTGGCCCAGTTGGA..........TGGAGCAACAACCTCCTAAG
>contig2
GGATGGATGTCTGAGCGGTT..........GAAAGAGTCGGTCTTGAAAA
>contig3
GGGAGAATAGTGCCAATGGT..........AGAACAATGGTCTCCAAAAC
>contig4
GTCTTTGCTTCGGTGGGAGT..........CTTGGTCTGCGGGTGGAGGT

Question 2:

Why can't I retrieve complete results or any results at all even after submitting my sequence and obtaining a Project ID?

Answer: There could be many reasons leading to this issue. The most important one could be that the sequence you submitted might not be complete, as some exons of trans-splicing genes could be missing. Moreover, considering the limited resources available for plant mitochondrial genomes, some unknown errors might not be detected during the early testing of IPMGA program. We encourage you to send your data to our email (lijingling@163.com), and we are willing to help you check for errors.

Question 3:

What is the role of the concatenated file in folder 2 of the result file, and can I use it to submit to NCBI?

Answer: If your submitted sequence only contains one contig, then the concatenated file and the annotated file in folder 1 are identical. However, please note that the concatenated file cannot be used for submitting to NCBI when your submitted FASTA file contains multiple contig sequences, because the concatenated file is pieced together by connecting the beginning and end of all submitted sequences and is not a real sequence in its mitogenome. Nevertheless, considering that different exons of some trans-splicing genes may be distributed on different contigs, we recommend using the concatenated file for gene sequence extraction because it would be more convenient.

Question 4:

What is the purpose of the other files except subfolders 01-08 of the resulting folder?

Answer: The other files are intermediate files generated during the program running process that are not generally needed by users. The original BLAST-based results are placed in the /results/ref/ folder and can be viewed if needed.