HIVDB GenBank Submission Tool

This tool can help you prepare aligned sequences, descriptive data, and sequence features for GenBank submission. It's designed for sanger sequencing or consensus sequence of NGS sequencing.

How to use the program

1. Prepare the sequence and meta data files
2. Run the script
3. Check BankIt files for submission

Prepare the sequence and meta data files

Two files are required:

1. sequence files in fasta format, file name is unaligned_sequences.fasta.
2. meta data file in csv format, file name is source_modifier.csv.

The meta data file should contain a column called Isolate, the Isolate is used as the unique ID for each sequence, it's also used in the fasta file to identify sequences.

The meta data should contain the columns below:

• Isolate
• Collection_date (formatted like '2024-01-01')
  • https://www.ncbi.nlm.nih.gov/WebSub/html/help/collection-date.html
• Country
  • Please check the name is correct, https://www.ncbi.nlm.nih.gov/genbank/collab/country/
• Isolation source
• Genes

Additional columns can also be added to the meta data file

• note
  • this column can contain deidentified patient code, treatment history in raw text

The GenBank document Preparing a Source Modifiers Table File for All Source Modifiers lists all the headers. If you want to add additional headers other than these headers listed above, please create a new issue and let us know.

Note: The program assumes the Organism is Human immunodeficiency virus 1, and the Host is Homo sapiens, which can be configured using script arguments.

Run the script

Save unaligned_sequences.fasta and source_modifier.csv under input_files folder.

make init
make

The program will do several things:

• check the uniqueness of Isolate
• align the sequence and get alignment information
• generate two BankIt files in bankit_files folder
  • bankit.fasta
  • features.txt

Exclude sequence

You can create a file input_files/aligned_ignore.csv to exclude some sequences with issues. The header of this file should contain Isolate and gene columns

Suggested sequences to be excluded:

• appearent sequencing error
• gap within a gene is too long
• too many stop codons
• too many untranslatable codons

Check BankIt files for submission

Before submitting your sequences and meta data to GenBank, you should double check the generated two files.

• check sequence quality file input_files/aligned_meta.csv
• check bankit.fasta
  • check the positions of stop codons are correct.
  • check the positions of deletions and gaps are correct.
  • run the HIVdb Program, compare the result of original sequence and the corresponding sequence in this file, the mutations should be the same.
• check features.txt file
  • check the start stop positions
  • check sequence with stop codons has misc feature
  • check sequence with gaps has gap information
  • check sequence with untranslatable codons has misc feature
  • check the gene name

Create BankIt submission (Updated at: 2023-12-06)

Prepare other information before submission

• Contact
• Reference
• Sequencing technology

Here is the BankIt tool.

Please see below are some issues you would encounter and how to resolve them:

• In Nucleotide tab, you should upload the bankit.fasta file

• In Features tab, you should upload the features.txt file

• Warning: There are one or more significant strings of NNNs (length>10). Please explain what the strings of internal NNNs represent

  • choose a region of estimated length between the sequenced regions based on an alignment to similar sequences or genome

• Submission Set/Bach

  • choose Pop set

• Warning: Terminal ends of the following sequence(s) are low quanlity (too many ambiguous bases) and have been trimmed.

  • choose or, click here to undo all trimming, and then click Continue to submit the original untrimmed sequences(s)
  • note: the BankIt may remove one nucleotide at the end because of ambiguity, which cause the last codon can not be translated
  • after your choice, at the end of the page, it will show bankit.fasta+(untrimmed+original)

• Sequence(s) and Definition Line(s), Molecule Type

  • if the sequences were isolated from plasma, choose genomic RNA

• Tab Sequencing Technology

  • if the sequences were not using NGS methods, please don't choose any of th options unassembled sequence reads, assembed sequences (consisting of two or more sequence reads).

Advanced usage

You can use your prefered alignment tool to prepare the aligned sequence and save in file input_files/aligned_meta.csv

Several columns are required

• Isolate
• gene
  • gene name
• insertions
  • insertion mutation list, for example (S255N_K)
• deletions
  • deletion position list
• stops
  • stop mutation list for example (E170*)
• gene_AA_length
  • gene amino acid sequence length
• gene_NA_length
  • gene nucleotide sequence length
• aligne_NA_length
  • nucleotide sequence length after alignment
• start_AA_pos
• start_NA_pos
• stop_AA_pos
• stop_NA_pos
• translatable
• untrans_reason
  • if the sequence is not translatable, please provide the reason
• aligned_NA
  • the aligned_NA should be the same length as gene_NA_length, and heading or tailing unsequenced positions should use . to indicate tehem.

Then you can use the command line to generate BankIt files, please refer to Makefile for how to use the scripts.

The issues you could meet and how to solve them

• 500 error
  • you may uploaded too many sequences, please try to split them to batches
• PUBMED ID not found
  • you need manually fill in the reference information, although it has PUBMED ID
• the sequences contains more than 50% N's
  • please remove these sequences