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Geneious Sequence Classifier User Manual

1. Unclassified 2 best matc 1 of 2 1 cy Abtei L com ai 100 100 Apteryx haastii Unknown1 cytochrome b Alignment amp Tree Database Sequence Name Overlap Identity Query Identity Taxonomy Apteryx haastii 34491 Mt 100 100 Apteryx haastii Apteryx haastii M3 cytoc 100 100 Apteryx haastii Apteryx haastii GS17 cyt 100 100 Apteryx haastii Apteryx haastii FT2922 c 100 100 Apteryx haastii Apteryx haastii FT2920 c 100 100 Apteryx haastii Apteryx haastii 8 23187 C 99 67 99 67 Apteryx haastii Figure 1 Results tables showing the Summary and Classification tables on the left and Iden tity tables on the right Identity tables are given for the overall result and for each gene in your database If any locus has an overlap identity below its specified cut off or no match can be found with its corresponding database sequence all loci for that sequence are excluded from the concatenated alignment and tree unless the option to include or ange results is turned on After building the concatenated alignment any concatenated sequence which has a combined weighted overlap identity below the general minimum overlap for multiple alignments will also be excluded e Save multiple alignment of all hits per query Creates a multiple alignment of all database hits for each query sequence Options for this alignment e g choice of alignment pro gram can be set by clicking the Alignment Options button By default only t
2. this character The sequence is classified based on the split sections of the taxonomies that are in common up to the point where they first differ e g a query that equally matches Acmophyle sahniana and Acmopyle pancheri will be classified as Acmophyle if the taxonomic separator is a space Minimum overlap identity to classify at lowest second or third taxonomic level These settings allow you to set thresholds for classifying to a particular taxonomic level The overlap identity of the best match to the query must be higher than the threshold for a particular taxonomic level for it to be classified to that level If the overlap identity of the best result is lower than the threshold for a particular taxonomic level then the result will be classified at a higher taxonomic level Note that a query may also be classified at a higher taxonomic level if multiple best matches are above the threshold but the multiple matches only have a higher taxonomic level in common as described above in the Minimum identity higher than next best result to classify section above The lowest taxonomic level does not necessarily have to be species it could subspecies locality or genus family etc The classifier will simply interpret the field selected for taxonomic classification according to the taxonomic separator and the lowest taxonomic level will be the final part of the taxonomy name E g if you choose the sequence name field t
3. Seguence Classifier for Geneious R8 or later Aaron Kennedy USDA APHIS PPO and Biomatters November 10 2014 geneioug Overview The Classify Seguences plugin classifies a guery seguence by aligning it against all seguences in a specified database and then choosing an appropriate taxon according to user specified identity levels Single or multiple genes can be used for classification and multiple query sequences can be run at once The plugin first performs pairwise global alignments with free end gaps between the query sequence and each sequence in the database When sequences from multiple genes are avail able for classification pairwise alignments for each gene are performed separately and these alignments are then concatenated if there are database sequences with the same name for each gene The Overlap Identity between your query and the database sequences is then used to de termine the likely taxon of your query sequence by picking the database sequence with the highest identity to the query The overlap identity is the pairwise identity in the region in com mon between the query and database sequence The sequence will only be classified if it meets a minimum overlap identity that the user specifies and if multiple database sequences have similar overlap identities then the query sequence will be classified to the taxonomic level that these sequences have in common It is also possible to set cutoffs for classifying seque
4. ferent minimum value for each locus Text View Info c Export table i Elri Classified 3 out of 3 sequences Summary Results for Unknown 1 Classification Frequency Frequency K Unclassified identity too low 1 33 33 Unknownl Alignment amp Tree Apteryx mantelli 1 33 33 Database Sequenc Overlap Identity Query Identity Taxonomy Loci Matched Apteryx haastii 1 33 33 Apteryx haastii S 100 35 79 Apteryx haastii 1 of 2 1 control Apteryx haastii G 10026 100 Apteryx haastii 2 of 2 control re Apteryx haastii 99 80 99 80 Apteryx haastii 2 of 2 control re Apteryx haastii M3 199 80 99 80 Apteryx haastii 2 of 2 control re Apteryx haastii G 99 80 99 80 Apteryx haastii 2 of 2 control re Apteryx haastii G 99 80 99 80 Apteryx haastii 2 of 2 control re Unknown1 control region Alignment 4 Tree Database Sequence Name Overlap Identity Query Identity Taxonomy Apteryx haastii 25792 O 100 92 11 Apteryx haastii Classifications Apteryx haastii S 23187 C 100 92 11 Apteryx haastii Query Sequence 4 Overlap Identity Classification Closest Segu Loci Matched Apteryx haastii modern c 100 100 Apteryx haastii Unknowni 100 Apteryx haa 2 best matc 1 of 2 1 co Apteryx haastii GS21 con 100 100 Apteryx haastii Unknown2 100 Apteryx ma 5 best matc 1 of 2 1 cy Apteryx haastii GS17 con 100 100 Apteryx haastii Unknown3 82 35
5. he green hits are included in the alignment see Highlight results in green above but red and orange hits can be included by checking these additional options e Save tree of all hits per query Builds a phylogenetic tree from the multiple alignment Parameters for tree building can be set by clicking the Tree Options button e Save alignments and trees in subfolder Creates a subfolder to save multiple alignments and trees into recommended when running multiple queries Note that when running the sequence classifier on a large number of query sequences checking addi tional results options can cause the result document to become quite large and or produce many result documents which can impact performance Interpreting Identity tables The top identity table shows the overall result when alignments from multiple loci are con catenated When multiple loci are used individual identity tables are also provided for each gene see Figure 2 The columns in these tables are as follows Database seguence name The name of the database seguence matching your guery Overlap Identity The percentage identity across the region where database and guery seguences overlap where data in end gap regions is ignored In the overall result table produced with multiple loci the Overlap Identity is a weighted average over the con stituent loci weighted according to the overlap length from each contributing locus Loci that could not be aligned to
6. he query sequence will not be classified if the overlap identity between the query and best database match is below this setting e Minimum identity higher than next best result to classify If other database se quences have an overlap identity within this value of the identity with the closest match then they will be considered to be equally good matches to the query In this instance if classify using taxonomy see below is not checked then Multiple matches will be returned as the classification If classify using taxonomy is checked then the query sequence will be classified to the taxonomic level that these database sequences have in common E g for the example dataset given above with classify using taxonomy checked if this value is set on 0 2 and the query sequence has 99 3 identity with Acmopyle pancheri and 99 2 identity with Acmopyle sahniana then it will only be classified as Acmopyle as the identities are within 0 2 of each other Classify using taxonomy from When this setting is checked you can classify using structured taxonomic levels rather than just the sequence name You can choose the Name Description Organism or Taxonomy fields of the database sequences to classify by The taxonomic levels will be delineated according to the Taxonomic Level Separator that you choose If a query sequence matches multiple database sequences with different taxonomies then the taxonomies are split via
7. hows the classification of each query sequence along with its identity to the closest database sequence Detailed Identity tables for each query can be produced by checking the option Create table of all hit similarities for each query sequence Note that if the sequence classifier is run on a single query sequence only Identity tables not Summary or Classification tables are produced and this option is greyed out Other result options can be configured as follows e Save pairwise alignments Produces a pairwise alignment document for each query and database sequence and saves them in a separate folder When a single query sequence is run double clicking on a particular row in the identity table will bring up the pairwise alignment for that result We recommend unchecking this option when running multiple queries as it can potentially produce a large number of alignment documents which can impact performance Highlight results in green with minimum overlap identity Results that exceed the over lap identity entered here will be colored in green in the identity tables Those below this value will be colored in red When multiple loci are used green results will instead be colored orange if all loci for that sequence are not green However the concatenated re sult will always be colored red if none of the loci exceed their green threshold no matter what the concatenated green threshold is e Per Locus Minimums Allows you to specify a dif
8. nces at various taxonomic levels For in stance if 95 is set as a minimum identity to classify at species level and the top match to the database has an overlap identity of 94 5 then the query will only be classified to genus level Thus you need to know the approximate levels of sequence identity within and between the different taxonomic levels of your database sequences in order to choose the correct settings for classification Setting up your database and query sequences Each sequence in the database must have a unique name and should be contained in a single folder in your Geneious database subfolders within this folder are allowed If only a single locus is used for classification the locus name is not required Formatting sequences when multiple loci are used If using multiple loci for classification the locus name must be appended to the sequence name with a specific delimiter in both the query and database sequences Geneious will use the sequence name before the delimiter to group database and query sequences from the same sample together For example example you may have 4 database sequences named Acmopyle pancheri matK Acmopyle panceri rbcL Acmopyle sahniana matK Acmopyle sahniana rbcL Your query sequences could be named Query1 matK and Query1 rbcL With multiple loci this is treated as a database containing 2 sequences called Acmopyle pancheri and Acmopyle sahniana and a single query se
9. o classify your sequences by with space as the separator and the sequence names are in the format Genus species locality then locality will be regarded as the lowest taxonomic level species as the second and genus as the third You may find it useful to perform a multiple alignment of your database sequences and then look at the Distances tab to get an idea of appropriate identity thresholds for differ ing taxonomic levels Multiple loci If you have seguences from multiple loci to use for classification check the Use Multiple Loci bor and put the delimiter you have used to separate the seguence name and locus name in the box underneath see Setting up your database section This option will concatenate individual pairwise alignments from different genes if the seguence names before the delimiter match When Replace end gaps with characters when concatenating alignments is checked then end gaps from individual pairwise alignments are treated as unknown characters in the con catenated alignment If this option is unchecked then the end gaps are regarded as real gaps in the concatenated alignment Results Sequence classifier results are displayed as tables in the sequence viewer see Figure 1 When multiple queries are run at once Summary and Classification tables are produced The Sum mary table shows how many query sequences are classified into each taxonomic group and the Classification table s
10. quence called Queryl When the analysis is run matK and rbcL sequences are aligned separately and then the alignments are concatenated according to their sequence name with used to replace any gaps between alignments Alignment options Options for the pairwise alignments between query and database sequences are specified in the Searching panel To choose the database you wish to align your query sequences to click the button next to Database folder and navigate to the folder containing your database sequences The Sensitivity setting specifies the parameters that Geneious uses to align the query and database sequences There is a trade off between how fast the search runs versus how sen sitive it is to finding distantly related matches A higher sensitivity will align queries that are more distantly related to the database so if you suspect your query sequence may be only dis tantly related to your database sequences or are not sure whether it matches you should use a higher sensitivity setting Classification settings From each pairwise alignment an Overlap Identity percentage is calculated for the region in common between two sequences where data in end gap regions is ignored The overlap identity is used to classify sequences by picking the database sequence with the highest overlap identity to the query sequence Parameters that can be set to classify sequences are as follows Minimum overlap identity to classify t
11. se sequence Results for Unknown1 Overall result Unknown1 li n ri Database Sequence Name Overlap Identity Query Identity Taxonomy Loci Matched 3 9 Apteryx haastii 2 1 control region Apteryx haastii GS17 100 100 Apteryx haastii 2 of 2 control region cytochrome b Apteryx haastii modern 99 80 99 80 Apteryx haastii 2 of 2 control region cytochrome b Apteryx haastii M3 99 80 99 80 Apteryx haastii 2 of 2 control region cytochrome b Apteryx haastii GS21 99 80 99 80 Apteryx haastii 2 of 2 control region cytochrome b Apteryx haastii GS10 99 80 99 80 Apteryx haastii 2 of 2 control region cytochrome b Unknown1 control region Alignment amp Tree Database Sequence Name Overlap Identity Query Identity Taxonomy Apteryx haastii 23187 Charleston1 contr 100 92 11 Apteryx haastii Apteryx haastii modern control region 100 100 Apteryx haastii Results for Apteryx haastii GS21 control region 100 100 Apteryx haastii each locus Apteryx haastii GS17 control region 100 100 Apteryx haastii Apteryx haastii GS10 control region 100 100 Apteryx haastii Unknown1 cytochrome b Alignment amp Tree Database Sequence Name Overlap Identity Query Identity Taxonomy Apteryx haastii FT2920 cytochrome b 100 100 Apteryx haastii Apteryx haastii 23187 Charlestonl cytoc 99 67 99 67 Apteryx haastii Apteryx haastii modern cytochrome b 99 67 99 67 Apteryx haastii Apteryx haastii GS21 c
12. the corresponding database sequence are assumed to have a completely mismatching overlap of length equal to the minimum overlap setting How ever if the database and query sequences do not contain some loci these loci are excluded from the weighted overlap identity without assuming there is a completely mismatching region Query Identity The percentage identity over the entire length of the query sequence where regions from the query that do not align to or are not covered by the database sequences are counted as mismatches Thus where the query identity is lower than the overlap identity it indicates that the query sequence extends outside the database se quence The overall query identity will be lowered if not all loci have a database match for a particular sequence Taxonomy Taken from the field you specified under Classify using taxonomy from Loci Matching Shows which loci in the database match the query when using multiple loci E g In the example in Figure 2 two loci are used cytochrome b and control re gion The database sequence Apteryx haastii S 25792 Oparara does not have a cytochrome b sequence so this entry reads 1 of 2 1 control region indicating that there are 2 query sequences that could potentially have matches as there is both control region and cytochrome b sequences for the query and 1 database sequence that could have poten tially matched as there is only a control region sequence for that databa
13. ytochrome b 99 67 99 67 Apteryx haastii Apteryx haastii GS11 cytochrome b 99 67 99 67 Apteryx haastii Apteryx haastii GS10 cytochrome b 99 67 99 67 Apteryx haastii Figure 2 Identity tables produced by the sequence classifier

Geneious Sequence Classifier User Manual

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