Submitting allele sequences to the GenBank NGSengine allele submission Sequin

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1 1 Submitting allele sequences to the GenBank 1 2 NGSengine allele submission NGSengine restrictions Allele names Generating the fasta file and feature table 2 3 Sequin Generating submitter information Preparing the submission Adding the FASTA sequence Adding the feature table Validation of the data Final steps for submission to GenBank 12 NGSengine can generate files that can be used to submit sequences of extended or new HLA alleles to GenBank. This is part of the process for submitting extended and new alleles. For a description of the full submission of an allele to the WHO HLA Nomenclature Committee for Factors of the HLA System, see This document describes step by step how an allele sequence can be submitted to GenBank using NGSengine and Sequin. After analysis NGSengine can generate the sequence information needed for a submission to GenBank. The data is saved in two files for each allele. One file is the DNA sequence in FASTA format, with extension.fsa. The second file contains the feature table, which describes the regions sequenced. This file has extension.tbl. The submission of alleles is only possible when for class I exon 2, intron 2 and exon 3, and for class II exon 2 are fully covered and phased. The submission involves the largest phased region containing these core regions. If e.g. phasing breaks before exon 7, then exon 7 is not included in the submission. In general there is only one phasing region ([R] 1), consequently the whole amplicon is included in submission.

2 The name of the allele to submit can be: - [Sample]_[Locus]_[Allelename]var (Sample01_HLA-B_B4050var) in case the submission involves exon mismatches - [Sample]_[Locus]_[Allelename]extended (Sample01_HLA-B_B4050extended) in case the submission is an allele extension - [Sample]_[Locus]_[Allelename] (Sample01_HLA-A_B ) in case no new nucleotides are involved in the submission - Right-click on the locus of the allele to submit - Select Prepare submission - A message appears that the action is successful or not successful Depending on the actual data, one of the two messages is shown: 1 2 When successful, the files are saved in the Submission folder in the project folder. An example with HLA-B alleles is shown in de figure below: Sequin is a stand-alone software tool developed by the NCBI for submitting sequences to the GenBank database. For details concerning Sequin installation and use, see Sequin can be used to prepare a submission to GenBank based on the following information: - Submitter information V

3 - The FSA file generated by NGSengine - The feature table in the TBL file generated by NGSengine The submitter information should be generated using Sequin, but can be saved for reuse for other submissions. Start Sequin: Click Start New Submission : Enter a tentative title. Click Next Page>> V

4 Enter the Author information. Click Next Page >> Click Next Page >> Select Click here to export a template and save the submitter information. Make sure the submitter information is entered either by entering as described, or by loading the submitter information by clicking on the Click here to import a template in the form below, and loading the submitter file. V

5 Click Next Page >> until the next form is on your screen. Select Use the normal submission dialog. Click Next. Select Single Sequence. Click Next Form >>. V

6 Select Import Nucleotide FASTA. Select the.fsa file of the allele to submit. The screen shows some details of the loaded FASTA sequence Click Next Page >>. V

7 Click Next Page >> Click Next Page >> V

8 Click Next Page >> Click Next Form >> V

9 Since no protein sequence has been entered, this warning is shown. The protein sequence is generated by NGSengine and should not be added. Click OK. Sequin now shows basic information about the allele submitted. In this case the submission concerns 2994 bases of B*35:03:01:01. V

10 The next steps add information about the gene such as the location of the exons and introns. Select File -> Open Select the feature table (.tbl file) generated by NGSengine for this allele. V

11 Sequin now has added the information of the feature table. One can inspect the location of all regions as well as the CDS, protein sequences etc. V

12 Sequin can validate whether the information is logical. This means that it checks, amongst others, whether the most basic information is present (like Organism ) and whether the description makes sense. The latter involves checking the splice sites, and the correct protein sequence. Select Search -> Validate The validation was successful. If Sequin reports warnings and/or errors, the sequence information is not complete or is not logical. This may be the case when splice site are affected in, for example, some null alleles. The last steps include preparing the file which should be send to the GenBank. Select File -> Prepare Submission. V

13 Save the.sqn file with a proper name. Follow the instructions in the warning by sending the file to gb-sub@ncbi.nlm.nih.gov. Once GenBank provides you with the proper accession number, you can continue with the submission at the IMGT/HLA website. Erik Rozemuller V