The sequencing-based typing tool of dbMHC: typing highly polymorphic g
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《核酸研究医学期刊》
National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Building 45, 8600 Rockville Pike, Bethesda, MD 20894, USA
* To whom correspondence should be addressed. Tel: +1 301 402 2781; Fax: +1 301 480 2484; Email: helmberg@ncbi.nlm.nih.gov
ABSTRACT
The dbMHC resource (http://www.ncbi.nlm.nih.gov/mhc/sbt.cgi?cmd=main) at the National Center for Biotechnology Information (NCBI) has developed an online tool for evaluating the allelic composition of sequencing-based typing (SBT) results of cDNA or genomic sequences. Whether the samples are heterozygous, haploid or a combination of the two, they can be compared with two up-to-date databases of all known alleles of several human leukocyte antigen (HLA) and killer cell immunoglobulin-like receptor (KIR) loci. The results of the submission are returned as a table of potential allele hits, along with the respective base changes and an interactive sequence viewer for close examination of the alignment.
INTRODUCTION
Direct sequencing of genomic DNA or cDNA is being used increasingly in routine diagnostic methods. For clinical procedures such as bone marrow transplants, detailed genotype information on both the recipient and the donor is required. Typically, five human leukocyte antigen (HLA) gene-coding regions of the major histocompatibility (MHC) complex are used for genotyping: HLA class I, HLA-A, HLA-B and HLA-C; and HLA class II, HLA-DRB1 and HLA-DQB1. In highly polymorphic genes such as HLAs, direct sequencing-based typing (SBT) is more efficient than traditional genetic testing methods, which are based on probe hybridization or amplification with sequence-specific primers. Sequencing strategies used in SBT differ between laboratories and can generate either heterozygous sequences, haploid sequences (after allele separation of the sample) or a combination of heterozygous and haploid sequences for each typed sample.
The dbMHC resource (http://www.ncbi.nlm.nih.gov/mhc/sbt.cgi?cmd=main) at the National Center for Biotechnology Information (NCBI) has developed an online tool for evaluating the allelic composition of SBT samples. Whether the samples are heterozygous, haploid or a combination of the two, they can be compared with two up-to-date databases of all known alleles of several HLA and killer cell immunoglobulin-like receptor (KIR) loci. HLA-related loci are based on the IMGT/HLA database (1), which contains sequences curated by the WHO allele nomenclature committee for HLA alleles. The allele database for KIR sequences is based on entries in GenBank and the IPD database (http://www.ebi.ac.uk/ipd/kir/index.html). The results of the submission are returned as a table of potential allele hits, along with the respective base changes and an interactive sequence viewer for close examination of the alignment.
To send feedback and suggestions for improvement, please Email the dbMHC team at: dbMHC@ncbi.nlm.nih.gov.
INPUT
Any number of sequences can be submitted; the number is indicated in the box provided (Figure 1). Sequences can be entered as simple text strings of bases or in FASTA format. All IUPAC nucleotide codes are valid; therefore, polymorphic positions of heterozygous sequences can also be represented.
Figure 1. The interface of the SBT resource. The page is divided into two frames. The top frame contains the submission form and displays the interpretation results; the bottom frame contains the interactive alignment viewer. In this example are three query sequences (Query 1–3), all testing the HLA-A locus from one sample. ‘Query 1’ and ‘Query 2’ are haploid, whereas ‘Query 3’ is heterozygous. One nucleotide mismatch is allowed between the query and matched sequences. Two allele combinations match perfectly; four allele combinations show one mismatch each. The blockwise display of the query sequences starts with ‘Query 1’. Nucleotides 1–270 of ‘Query 1’ are part of exon 2. The bottom frame shows the three query sequences aligned to the reference sequence A*010101. The mismatch for the allele combination A*0102–A0103 at position 363 (A<>A,G) is highlighted. The nucleotide ‘A’ in the heterozygous ‘Query 3’ does not match with the combination A,G of A*0102–A0103.
By default, sequences are analyzed as heterozygote allele combinations. If a sequence is haploid, this should be indicated by using the haploid check box, thus avoiding the assumption that the submission is heterozygous. Sometimes an allele will be segregated, i.e. covering a long region that has been sequenced in two or more fragments, e.g. an amplification that spans exon 2, intron 2 and exon 3, but only exons 2 and 3 have been sequenced. In this case, it is essential to know about the haplotypic link between exons 2 and 3. This can be specified using the linked property, for either strand one or strand two (Figure 1). Once the submisson information has been added, the final step is to select the locus that has been sequenced and enter the maximum number of nucleotide mismatches allowed between the submitted sequence and the alleles in the database. A gapped interpretation can also be selected, should the submitted sequence(s) contain a longer deletion than has thus far been observed at this locus.
Algorithm
Queries to the SBT Tool are processed in several steps. The first involves a BLAST-based (2,3) search on a custom-made BLAST database that contains the allele sequences of each locus. Each sequence is split into blocks of untranslated regions (UTRs), exons and introns. In the second step, the block ends are fine-tuned to ensure a complete alignment. This is important when the query sequences have as-yet-undescribed polymorphisms occurring around block boundaries. An alignment consisting of the best matches between blocks from the allele database and blocks of query sequences is created against a reference sequence. In the next step, all alleles that have nucleotide mismatches exceeding the user-defined cutoff are discarded. Insertions/deletions are counted as one mismatch. In submissions that contain several sequences, subsequent queries are run against only those sequences that were not discarded because of mismatches. If the default heterozygous analysis is requested, all of the remaining allele matches are combined at the end of the process to display a heterozygous allele.
OUTPUT
Allele combinations that match the submitted sequences within the user-defined cutoff are listed in a table. Nucleotide mismatches, insertions and deletions between the submitted sequences and any derived allele combination are listed according to their positions within the alignment and the nature of the mismatch. Each sequence submitted is split up according to its aligned UTR/exon/intron components. The blocks are listed according to their boundary positions within each sequence.
Heterozygous sequences that contain a deletion in one allele, which generates a base shift with almost continuous polymorphic positions after the deletion, can be correctly diagnosed, although the user should check to ensure that the electropherogram has been read correctly.
Interactive alignment viewer
Specifiying the locus that query sequence(s) will be BLASTed against is part of the submisison process (Figure 1). As soon as the user does this, the interactive alignment viewer is loaded into the bottom frame of the Web page. It displays the reference sequence with the aligned query sequences. To this can be added, as the user wishes, the matched alleles listed in the table of allele combinations and/or as many other alleles from the specified locus as desired (Figure 1). The alignment can be navigated by scrolling forward and backward, by selecting a distinct exon/intron/UTR from the block display or by specifying a position within the alignment. The nucleotide mismatches listed in the results table link directly to the position of the mismatch in the alignment viewer.
The various function buttons on the alignment viewer can be used to manipulate the alignment in the followng ways: translate the nucleotide sequence into protein, change the reference sequence, highlight single nucleotide polymorphism (SNP) positions, display sequences in FASTA format and download complete or partial alignments of sequences displayed.
DOWNLOAD OPTIONS AND FURTHER ANALYSIS
The results of the SBT tool—the tabulated allele matches, the sequence blocks used and/or the alignment—can be downloaded as a text file.
If there is a protein structure available for the locus selected, it can be viewed using the NCBI Cn3D viewer (4,5). All haploid sequence submissions are translated into proteins. When these sequences represent new alleles with mutations that lead to non-synonymous amino acid changes, those positions can be highlighted on the protein structure in the Cn3D viewer, and the file can be downloaded. This feature also allows the display of any amino acid differences between any subset of alleles.
ALLELE DATABASES
Currently, the databases are restricted to classical HLA and KIR genes, but we plan to expand the list of loci in the future. Allele databases populated from non-curated external sources (such as the KIR database) are updated periodically by an automated GenBank search. Sequences that match any existing allele with a maximum of 30 nt differences are included; some sequences may be entered by hand. Such sequences link back to the source GenBank record via the Accession number. (HLA loci, which already undergo high-quality manual curation, are not updated using GenBank sequences.)
REFERENCES
Robinson,J., Waller,M.J., Parham,P., de Groot,N., Bontrop,R., Kennedy,L.J., Stoehr,P. and Marsh,S.G. ( (2003) ) IMGT/HLA and IMGT/MHC: sequence databases for the study of the major histocompatibility complex. Nucleic Acids Res., , 31, , 311–314.
Altschul,S.F., Gish,W., Miller,W., Myers,E.W. and Lipman,D.J. ( (1990) ) Basic local alignment search tool. J. Mol. Biol., , 215, , 403–410.
Altschul,S.F., Madden,T.L., Schaffer,A.A., Zhang,J., Zhang,Z., Miller,W. and Lipman,D.J. ( (1997) ) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res., , 25, , 3389–3402.
Hogue,C.W. ( (1997) ) Cn3D: a new generation of three-dimensional molecular structure viewer. Trends Biochem. Sci., , 22, , 314–316.
Wang,Y., Geer,L.Y., Chappey,C., Kans,J.A. and Bryant,S.H. ( (2000) ) Cn3D: sequence and structure views for Entrez. Trends Biochem. Sci., , 25, , 300–302.(Wolfgang Helmberg*, Raymond Dunivin and )
* To whom correspondence should be addressed. Tel: +1 301 402 2781; Fax: +1 301 480 2484; Email: helmberg@ncbi.nlm.nih.gov
ABSTRACT
The dbMHC resource (http://www.ncbi.nlm.nih.gov/mhc/sbt.cgi?cmd=main) at the National Center for Biotechnology Information (NCBI) has developed an online tool for evaluating the allelic composition of sequencing-based typing (SBT) results of cDNA or genomic sequences. Whether the samples are heterozygous, haploid or a combination of the two, they can be compared with two up-to-date databases of all known alleles of several human leukocyte antigen (HLA) and killer cell immunoglobulin-like receptor (KIR) loci. The results of the submission are returned as a table of potential allele hits, along with the respective base changes and an interactive sequence viewer for close examination of the alignment.
INTRODUCTION
Direct sequencing of genomic DNA or cDNA is being used increasingly in routine diagnostic methods. For clinical procedures such as bone marrow transplants, detailed genotype information on both the recipient and the donor is required. Typically, five human leukocyte antigen (HLA) gene-coding regions of the major histocompatibility (MHC) complex are used for genotyping: HLA class I, HLA-A, HLA-B and HLA-C; and HLA class II, HLA-DRB1 and HLA-DQB1. In highly polymorphic genes such as HLAs, direct sequencing-based typing (SBT) is more efficient than traditional genetic testing methods, which are based on probe hybridization or amplification with sequence-specific primers. Sequencing strategies used in SBT differ between laboratories and can generate either heterozygous sequences, haploid sequences (after allele separation of the sample) or a combination of heterozygous and haploid sequences for each typed sample.
The dbMHC resource (http://www.ncbi.nlm.nih.gov/mhc/sbt.cgi?cmd=main) at the National Center for Biotechnology Information (NCBI) has developed an online tool for evaluating the allelic composition of SBT samples. Whether the samples are heterozygous, haploid or a combination of the two, they can be compared with two up-to-date databases of all known alleles of several HLA and killer cell immunoglobulin-like receptor (KIR) loci. HLA-related loci are based on the IMGT/HLA database (1), which contains sequences curated by the WHO allele nomenclature committee for HLA alleles. The allele database for KIR sequences is based on entries in GenBank and the IPD database (http://www.ebi.ac.uk/ipd/kir/index.html). The results of the submission are returned as a table of potential allele hits, along with the respective base changes and an interactive sequence viewer for close examination of the alignment.
To send feedback and suggestions for improvement, please Email the dbMHC team at: dbMHC@ncbi.nlm.nih.gov.
INPUT
Any number of sequences can be submitted; the number is indicated in the box provided (Figure 1). Sequences can be entered as simple text strings of bases or in FASTA format. All IUPAC nucleotide codes are valid; therefore, polymorphic positions of heterozygous sequences can also be represented.
Figure 1. The interface of the SBT resource. The page is divided into two frames. The top frame contains the submission form and displays the interpretation results; the bottom frame contains the interactive alignment viewer. In this example are three query sequences (Query 1–3), all testing the HLA-A locus from one sample. ‘Query 1’ and ‘Query 2’ are haploid, whereas ‘Query 3’ is heterozygous. One nucleotide mismatch is allowed between the query and matched sequences. Two allele combinations match perfectly; four allele combinations show one mismatch each. The blockwise display of the query sequences starts with ‘Query 1’. Nucleotides 1–270 of ‘Query 1’ are part of exon 2. The bottom frame shows the three query sequences aligned to the reference sequence A*010101. The mismatch for the allele combination A*0102–A0103 at position 363 (A<>A,G) is highlighted. The nucleotide ‘A’ in the heterozygous ‘Query 3’ does not match with the combination A,G of A*0102–A0103.
By default, sequences are analyzed as heterozygote allele combinations. If a sequence is haploid, this should be indicated by using the haploid check box, thus avoiding the assumption that the submission is heterozygous. Sometimes an allele will be segregated, i.e. covering a long region that has been sequenced in two or more fragments, e.g. an amplification that spans exon 2, intron 2 and exon 3, but only exons 2 and 3 have been sequenced. In this case, it is essential to know about the haplotypic link between exons 2 and 3. This can be specified using the linked property, for either strand one or strand two (Figure 1). Once the submisson information has been added, the final step is to select the locus that has been sequenced and enter the maximum number of nucleotide mismatches allowed between the submitted sequence and the alleles in the database. A gapped interpretation can also be selected, should the submitted sequence(s) contain a longer deletion than has thus far been observed at this locus.
Algorithm
Queries to the SBT Tool are processed in several steps. The first involves a BLAST-based (2,3) search on a custom-made BLAST database that contains the allele sequences of each locus. Each sequence is split into blocks of untranslated regions (UTRs), exons and introns. In the second step, the block ends are fine-tuned to ensure a complete alignment. This is important when the query sequences have as-yet-undescribed polymorphisms occurring around block boundaries. An alignment consisting of the best matches between blocks from the allele database and blocks of query sequences is created against a reference sequence. In the next step, all alleles that have nucleotide mismatches exceeding the user-defined cutoff are discarded. Insertions/deletions are counted as one mismatch. In submissions that contain several sequences, subsequent queries are run against only those sequences that were not discarded because of mismatches. If the default heterozygous analysis is requested, all of the remaining allele matches are combined at the end of the process to display a heterozygous allele.
OUTPUT
Allele combinations that match the submitted sequences within the user-defined cutoff are listed in a table. Nucleotide mismatches, insertions and deletions between the submitted sequences and any derived allele combination are listed according to their positions within the alignment and the nature of the mismatch. Each sequence submitted is split up according to its aligned UTR/exon/intron components. The blocks are listed according to their boundary positions within each sequence.
Heterozygous sequences that contain a deletion in one allele, which generates a base shift with almost continuous polymorphic positions after the deletion, can be correctly diagnosed, although the user should check to ensure that the electropherogram has been read correctly.
Interactive alignment viewer
Specifiying the locus that query sequence(s) will be BLASTed against is part of the submisison process (Figure 1). As soon as the user does this, the interactive alignment viewer is loaded into the bottom frame of the Web page. It displays the reference sequence with the aligned query sequences. To this can be added, as the user wishes, the matched alleles listed in the table of allele combinations and/or as many other alleles from the specified locus as desired (Figure 1). The alignment can be navigated by scrolling forward and backward, by selecting a distinct exon/intron/UTR from the block display or by specifying a position within the alignment. The nucleotide mismatches listed in the results table link directly to the position of the mismatch in the alignment viewer.
The various function buttons on the alignment viewer can be used to manipulate the alignment in the followng ways: translate the nucleotide sequence into protein, change the reference sequence, highlight single nucleotide polymorphism (SNP) positions, display sequences in FASTA format and download complete or partial alignments of sequences displayed.
DOWNLOAD OPTIONS AND FURTHER ANALYSIS
The results of the SBT tool—the tabulated allele matches, the sequence blocks used and/or the alignment—can be downloaded as a text file.
If there is a protein structure available for the locus selected, it can be viewed using the NCBI Cn3D viewer (4,5). All haploid sequence submissions are translated into proteins. When these sequences represent new alleles with mutations that lead to non-synonymous amino acid changes, those positions can be highlighted on the protein structure in the Cn3D viewer, and the file can be downloaded. This feature also allows the display of any amino acid differences between any subset of alleles.
ALLELE DATABASES
Currently, the databases are restricted to classical HLA and KIR genes, but we plan to expand the list of loci in the future. Allele databases populated from non-curated external sources (such as the KIR database) are updated periodically by an automated GenBank search. Sequences that match any existing allele with a maximum of 30 nt differences are included; some sequences may be entered by hand. Such sequences link back to the source GenBank record via the Accession number. (HLA loci, which already undergo high-quality manual curation, are not updated using GenBank sequences.)
REFERENCES
Robinson,J., Waller,M.J., Parham,P., de Groot,N., Bontrop,R., Kennedy,L.J., Stoehr,P. and Marsh,S.G. ( (2003) ) IMGT/HLA and IMGT/MHC: sequence databases for the study of the major histocompatibility complex. Nucleic Acids Res., , 31, , 311–314.
Altschul,S.F., Gish,W., Miller,W., Myers,E.W. and Lipman,D.J. ( (1990) ) Basic local alignment search tool. J. Mol. Biol., , 215, , 403–410.
Altschul,S.F., Madden,T.L., Schaffer,A.A., Zhang,J., Zhang,Z., Miller,W. and Lipman,D.J. ( (1997) ) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res., , 25, , 3389–3402.
Hogue,C.W. ( (1997) ) Cn3D: a new generation of three-dimensional molecular structure viewer. Trends Biochem. Sci., , 22, , 314–316.
Wang,Y., Geer,L.Y., Chappey,C., Kans,J.A. and Bryant,S.H. ( (2000) ) Cn3D: sequence and structure views for Entrez. Trends Biochem. Sci., , 25, , 300–302.(Wolfgang Helmberg*, Raymond Dunivin and )