Design and Annotation Files

Transcription

1 Design and Annotation Files Release Notes SeqCap EZ Exome Target Enrichment System The design and annotation files provide information about genomic regions covered by the capture probes and the genes included in these regions. This document covers the following products: SeqCap EZ MedExome Enrichment Kit SeqCap EZ MedExome Plus Enrichment Kit SeqCap EZ Human Exome Library v3.0 SeqCap EZ Exome Plus Library SeqCap EZ Exome +UTR Library SeqCap EZ Human Exome Library v2.0 The SeqCap EZ MedExome Enrichment Kits use genome coordinates from the UCSC human genome build hg38. Files in hg19 are also provided. All other products listed in this document use genome coordinates from the UCSC human genome build hg19. Each SeqCap EZ Exome system contains files for viewing the design and annotations. Notes about these file formats: <File_name >.bed: BED files are plain text, tab-delimited files which list genomic coordinates. These files may be used to explore design targets and to assess capture performance within the targeted regions. The first 3 columns are chromosome or sequence name, target start (0-based), and target end (1- based). Some BED files delivered from Roche NimbleGen include two tracks. In this case, the tracks should be split into separate files prior to use in analysis. A BED file can be displayed as a custom annotation track using the UCSC Genome browser ( and can also be opened using SignalMap software (Roche NimbleGen, <File_name >.gff: GFF files are similar to BED files but with a different set of tab-delimited columns. GFF files are included for older designs from Roche NimbleGen. GFF files are not included with SeqCap EZ MedExome Enrichment Kits. For life science research only. Not for use in diagnostic procedures.

2 SeqCap EZ MedExome Enrichment Kit Design and annotation files were designed for use with the following products: SeqCap EZ MedExome Enrichment Kit, 4 Reactions SeqCap EZ MedExome Enrichment Kit, 48 Reactions SeqCap EZ MedExome Enrichment Kit, 384 Reactions The SeqCap EZ MedExome Enrichment Kit was designed based on the following databases: CCDS 17 RefSeq CDS August 2014 Ensembl 76 CDS (biotype filtered) VEGA 56 CDS GENCODE 20 CDS mirbase 21 The design also includes coverage for regions defined as medically relevant, including: GeneTests CDS (excluding mitochondrial genes) ClinVar (likely pathogenic or pathogenic) coding and non-coding variants Coding sequence from the set of ~4600 genes identified by the consortium of the Emory Genetics Lab, Harvard Laboratory of Molecular Medicine, and Children's Hospital of Philadelphia (CHOP) Additional regions deemed as medically relevant based on customer input File Descriptions Design files delivered with a SeqCap EZ MedExome Enrichment Kit: MedExome_hg38_capture_targets.bed: This file, in BED format, contains capture target intervals along with associated annotation IDs. The coordinates listed here correspond to locations where capture probes were actually designed and placed. MedExome_hg38_empirical_targets.bed: The empirical targets BED file shows regions covered by at least 20X across seventy-five percent of multiple captures given 6 gigabases each (about 60 million reads). This is a new type of target BED file that is being provided to show regions of reproducible coverage. If your protocol differs from the most recent version of the SeqCap EZ SR User's Guide, your coverage results may also vary. In this case, padding added to the capture targets appropriate for your typical insert size can provide an alternative target for mapping and performance assessment. For further guidance or clarification, contact Roche Technical Support ( MedExome_hg19_capture_targets.bed: MedExome capture target intervals converted to UCSC human genome build hg19 using the NCBI Genome Remapping Service. MedExome_hg19_empirical_targets.bed: MedExome empirical target intervals converted to UCSC Human Genome assembly build hg19 using the NCBI Genome Remapping Service. 2 NimbleGen SeqCap EZ Exome Library v2.0 Design and Annotation Files

3 SeqCap EZ MedExome Plus Enrichment Kit SeqCap EZ MedExome Plus Enrichment Kit, 48 Reactions The SeqCap EZ MedExome Plus Enrichment Kit is based on the enrichment targets from the SeqCap EZ MedExome Enrichment Kit plus up to 200 Mb of your custom design. File Descriptions Design files delivered with a SeqCap EZ MedExome Plus Enrichment Kit: Capture target regions obtained by merging MedExome capture targets and the capture targets of your custom design ( DESIGN ): MedEx_DESIGN_capture_targets.bed SeqCap EZ MedExome Plus Enrichment Kit design files: o MedExome_BUILD_capture_targets.bed: probe footprint for MedExome. o MedExome_BUILD_empirical_targets.bed: empirical target for MedExome. where BUILD is either hg19 or hg38 to match the genome assembly of your custom design. Design files associated with your custom design: o DESIGN_primary_targets.bed: primary target intervals for custom design. o DESIGN_capture_targets.bed: probe footprint for custom design o DESIGN_coverage_summary.txt: probe coverage summary table for custom design. o Note that when an older custom design is used in SeqCap EZ MedExome Plus Enrichment Kit, the custom design files may alternatively include a GFF file and a two-track BED file instead of two separate BED files. NimbleGen SeqCap EZ Exome Library v2.0 Design and Annotation Files 3

4 SeqCap EZ Human Exome Library v3.0 Design and annotation files were designed for use with the following products: SeqCap EZ Human Exome Library v3.0, 4 Reactions (Catalog No ) SeqCap EZ Human Exome Library v3.0, 48 Reactions (Catalog No ) The SeqCap EZ Human Exome Library v3.0 product was designed based on the following databases: NCBI Reference Sequence (RefSeq) RefGene from UCSC (GRCh37_CDS_ ) CCDS.2 from NCBI GRCh37_ Vega (GRCh37_CDS_42) Gencode(GRCh37_CDS_v3C) Ensembl (GRCh37_CDS_v63) mirnas from mirbase (version 16) mirnas from snornabase (version 3) Customer inputs For RefSeq genes, only transcripts with an NM_ prefix were selected, and only protein coding parts of the transcripts were targeted. For exons that are smaller than 100 bp, Roche NimbleGen extended the target region to 100 bp. More than two million long oligonucleotide DNA probes were designed to capture the target regions. Because the flanking regions of some coding exons and mirnas are also covered by probes, the total size of the regions covered by probes is 64 Mb. File Descriptions The folder contains these three files: SeqCap_EZ_Exome_v3_primary.bed: This file contains primary target intervals along with associated annotation IDs in BED format. SeqCap_EZ_Exome_v3_capture.bed: This file contains capture target intervals along with associated annotation IDs in BED format. The coordinates listed here correspond to locations where capture probes were actually designed and placed. If an exon was originally targeted for capture, but probes could not be placed in that region (for example, due to highly repetitive sequences), then the coordinates would be included in the SeqCap_EZ_Exome_v3_primary.bed file but not included in the SeqCap_EZ_Exome_v3_capture.bed file. Annotations provided in this file assume a 100 bp theoretical padding surrounding the capture targets. Annotation provided in the BED file is derived from Ensembl Genes (version 64) and includes IDs for RefSeq, CCDS, Ensembl, Vega, mirbase along with the associated gene_name for each interval. SeqCap_EZ_Exome_v3.gff: This file contains both primary target and capture target intervals in GFF format. 4 NimbleGen SeqCap EZ Exome Library v2.0 Design and Annotation Files

5 SeqCap EZ Exome +UTR Library Design and annotation files were designed for use with the following products: SeqCap EZ Exome +UTR Library, 4 Reactions (Catalog No ) SeqCap EZ Exome +UTR Library, 48 Reactions (Catalog No ) The SeqCap EZ Exome +UTR Library product is based on the same coding exon sources from SeqCap EZ Human Exome Library v3.0 (described elsewhere in this document) with expanded coverage of 5 - and 3 -untranslated regions (UTRs) from the following sources: NCBI Reference Sequence (RefSeq) refgene table from UCSC GRCh37/hg19 March 2012 Ensembl (GRCh37 v64) The folder contains these files: _HG19_ExomeV3_UTR_EZ_HX1_primary_annotated.bed: This file contains primary target intervals along with associated annotation IDs in BED format _HG19_ExomeV3_UTR_EZ_HX1_capture_annotated.bed: This file contains capture target intervals along with associated annotation IDs in BED format. The coordinates listed here correspond to locations where capture probes were actually designed and placed. SeqCap EZ Exome Plus Library Design and annotation files were designed for use with the following Roche NimbleGen products: SeqCap EZ Exome Plus Library, 12 Reactions (Catalog No ) SeqCap EZ Exome Plus Library, 48 Reactions (Catalog No ) SeqCap EZ Exome Plus Library, 96 Reactions (Catalog No ) The SeqCap EZ Exome Plus Library product is based on the gene sources from SeqCap EZ Human Exome Library v3.0 plus up to 200 Mb of your custom design. Refer to SeqCap EZ Human Exome Library v3.0 for information. NimbleGen SeqCap EZ Exome Library v2.0 Design and Annotation Files 5

6 SeqCap EZ Human Exome Library v2.0 Design and annotation files were designed for use with the following products: SeqCap EZ Human Exome Library v2.0, 4 Reactions (Catalog No ) SeqCap EZ Human Exome Library v2.0, 48 Reactions (Catalog No ) The SeqCap EZ Human Exome Library v2.0 product was designed from the following databases: NCBI Reference Sequence (RefSeq) RefGene from UCSC (January 2010) CCDS from NCBI (September 2009) mirnas from mirbase (version 14, September 2009) Customer inputs For RefSeq genes, only transcripts with an NM_ prefix were selected, and only protein coding parts of the transcripts were targeted. For exons that are smaller than 100 bp, Roche NimbleGen extended the target region to 100 bp. The total size of the target regions is 36.5 Mb. Roche NimbleGen selected 2.1 million long oligo probes to cover the target regions. Because some flanking regions are also covered by probes, the total size of regions covered by probes is 44.1 Mb, larger than the initial target regions. In the file descriptions provided in the next section, target regions refer to the 36.5 Mb targets selected from various databases, and probe-covered regions refer to the 44.1 Mb regions covered by long oligo capture probes. File Descriptions The Target_Regions folder contains these two files: SeqCap_EZ_Exome_v2.gff: There are two tracks in this.gff file. The primary_target_region track displays the 36.5 Mb target regions, and the capture_target track displays the 44.1 Mb probe-covered regions. The GFF files can be opened using SignalMap software (Roche NimbleGen, SeqCap_EZ_Exome_v2.bed: There are two tracks in this.bed file. The target_region track displays the 36.5 Mb target regions, and the tiled_region track displays the 44.1 Mb probe-covered regions. The Annotations folder contains these four files: SeqCap_EZ_Exome_v2_annotations.xls: This Microsoft Excel file lists the genes and mirnas that are targeted by the design. There are three worksheets in the file, listing RefSeq genes, mirna genes, and other genes. The other genes include customer provided genes (using the UCSC Genes database) and CCDS genes that are not in the RefSeq database. Most column headers are self-explanatory. Two of the columns provide information about how well the specific exon target is covered by the capture probes: ARRAY COVERAGE. Percentage of target base covered by probes. Be aware that a region not covered by probes can still be captured if its neighboring regions are 6 NimbleGen SeqCap EZ Exome Library v2.0 Design and Annotation Files

7 covered by probes. Refer to the following description of the ARRAY COVERAGE W 100BP EXTENSION column for more information. ARRAY COVERAGE W 100BP EXTENSION. Percentage of target base covered by probes or located within 100 bp to one or more probes. Because the DNA fragments captured by the SeqCap EZ Human Exome Library are generally greater than 200 bp, sequencing results typically show sufficient coverage of bp flanking regions at both sides of a region targeted by probes. Therefore, coverage with 100 bp extension is a better estimate of how much of the target region will receive sequence coverage. SeqCap_EZ_Exome_v2_RefSeq.gff, SeqCap_EZ_Exome_v2_miRNA.gff, SeqCap_EZ_Exome_v2_other.gff: There is a single track in each of these.gff files. The track lists the original coordinates of the exon targets as determined from the various databases. The other genes include customer provided genes (using UCSC Genes database) and CCDS genes that are not in the RefSeq database. These files can be loaded into the SignalMap software, and each vertical bar represents one exon target. When using SignalMap software, move the cursor over each exon to display the accession number/sequence identifier. NimbleGen SeqCap EZ Exome Library v2.0 Design and Annotation Files 7

8 Technical Support If you have questions, please contact your local Roche Technical Support. Go to for contact information /15 For life science research only. Not for use in diagnostic procedures. NIMBLEGEN and SEQCAP are trademarks of Roche. Published by Roche NimbleGen, Inc. 500 S. Rosa Rd Madison, WI USA Other brands or product names are trademarks of their respective holders Roche NimbleGen, Inc. All rights reserved.