De novo assembly of the pepper transcriptome (Capsicum annuum): a benchmark for in silico discovery of SNPs, SSRs and candidate genes

Transcription

1 Additional_file Ashrafi_et_al_0_Pepper_Annotation_Supp_0070.docx A Microsoft-Word 007 file with figures comparing the results of BlastGO for GeneChip (Sanger-EST) and transcriptome assemlies of pepper as well as the IGA transcriptome assemly procedure flow chart. 7 De novo assemly of the pepper transcriptome (Capsicum annuum): a enchmark for in silico discovery of SNPs, SSRs and candidate genes AUTHORS: Hamid Ashrafi, Theresa Hill, Kevin Stoffel, Alexander Kozik, Jiqiang Yao, Seastian Reyes Chin-Wo and Allen Van Deynze

2 a Supplement Figure. Distriution of E-Values of BLASTX of a) the Sanger- EST unigenes ) IGA transcriptome contigs

3 a Supplement Figure. Percent Similarity of assemly sequences with sequences in the GenBank a) Sanger-EST unigenes ) IGA transcriptome contigs. Similarity is computed of each query-hot pair as the sum of similarity values for all matching HSPs

4 a Supplement Figure. Length vs numer of sequences in a) Sanger-EST unigenes ) IGA transcriptome contigs.

5 a Supplement Figure. High-scoring segment pairs (HSP) per sequence coverage a) Sanger-EST unigenes ) IGA transcriptome contigs.

6 a Supplement Figure. Evidence code distriution sequences depicts the inference aout the annotation. For instance IEA is inferred from electronic assay, or IDA inferred from direct assay. a) Sanger-EST unigenes ) IGA transcriptome contigs. Once mapping has een completed, the user can check the distriution of evidence codes in the recovered GO terms and the original dataase sources of annotations. These charts give an indication of suitale values for BG annotation parameters. For example, when a good overall level of sequence similarity is otained for the dataset, the default annotation cutoff value could e raised to improve annotation accuracy. Similarly, if evidence code charts indicate a low representation of experimentally derived GOs, the user might choose to increase the weight given to annotations. After the final annotation step, new charts show the distriution of annotated sequences, electronic the numer of GOs per sequence, the numer of sequences per GO, and the distriution of annotations per GO level, which jointly provide a general overview of the performance of the annotation procedure.

7 a Supplement Figure. Evidence code distriution for BLAST hits depicts the inference aout the annotation. For instance IEA is inferred from electronic assay, or IDA inferred from direct assay. a) Sanger-EST unigenes ) IGA transcriptome contigs.

8 a Supplement Figure 7. Numer of high similarity pairs per BLAST hit a) Sanger-EST unigenes ) IGA transcriptome contigs.

9 a Supplement Figure 8. Dataase resources that were used for mapping step of BLASTGO a) Sanger-EST unigenes ) IGA transcriptome contigs.

10 Numer of Contigs Numer of Contigs a Numer of GO terms Numer of GO terms Supplement Figure 9. Numer of GO terms per contigs. a) On average (weighted average) GO terms was mapped to 9,9 (%) contigs of Sanger-EST assemly. ) on average (weighted average) etween GO terms was mapped to 7,000 (0%) contigs of IGA transcriptome assemly.

11 a Supplement Figure 0. Numer of annotations at each GO level. P for Biological Processes, F for Molecular Function and C stands for Cellular components. a) Sanger-EST unigenes ) IGA transcriptome contigs.

12 Biological Function Biological Processe. a Direct Go Counts of Biological Processes Numer of Sequences Cellular process Metaolic process Response to stimulus Biological regulation Localization Developmental process Multicellular organismal process Cellular component organization Signaling Cellular component iogenesis Reproduction Multi-organism process Cell wall organization or iogenesis Growth Death Immune system process Caron utilization Rhythmic process Cell proliferation Viral reproduction Cell killing Locomotion Biological adhesion Nitrogen utilization Pigmentation Transferase activity Nucleotide inding Ion inding Hydrolase activity Protein inding Nucleic acid inding Oxidoreductase activity Transmemrane transporter activity Sustrate-specific transporter activity Cofactor inding Ligase activity Sequence-specific DNA inding TF activity Lyase activity Structural constituent of riosome Signal transducer activity Tetrapyrrole inding Isomerase activity Vitamin inding Carohydrate inding Lipid inding Enzyme inhiitor activity Metal cluster inding Peroxidase activity Caroxylic acid inding Nucleoside-triphosphatase regulator activity Enzyme activator activity Amine inding Direct GO Counts of Molecular Functions Numer of Sequences

13 Cellular Component c Direct Go Counts of Cellular Components Intracellular Iintracellular part Memrane Intracellular organelle part Memrane part Organelle memrane Organelle lumen Memrane-ounded vesicle Envelope Organelle envelope Endomemrane system External encapsulating structure Organelle sucompartment Network of nuclear outer & ER memranes Serine/threonine phosphatase complex Golgi memrane Vesicle memrane Intrinsic to organelle memrane Cell fraction Protein histidine kinase complex Beta-galactosidase complex Cell surface Extracellular matrix External encapsulating structure part Extracellular space Apical part of cell Periplasmic space Receptor complex Numer of Sequnces Supplement Figure. Direct GO count graphs depicting, a) Biological processes ) Cellular components and c) Molecular functions in the Sanger-EST assemly. 7 8

14 Molecular Function Biological Process a Cellular process Metaolic process Response to stimulus Biological regulation Localization Multicellular organismal process Developmental process Cellular component organization Signaling Reproduction Cellular component iogenesis Multi-organism process Death Growth Cell wall organization or iogenesis Immune system process Rhythmic process Caron utilization Cell proliferation Locomotion Viral reproduction Cell killing Biological adhesion Pigmentation Nitrogen utilization Sulfur utilization Nucleic acid inding Transferase activity Nucleotide inding Protein inding Hydrolase activity Ion inding Oxidoreductase activity Transmemrane transporter activity Sequence-specific DNA inding TF activity Sustrate-specific transporter activity Signal transducer activity Cofactor inding Ligase activity Lyase activity Tetrapyrrole inding Structural constituent of riosome Isomerase activity Carohydrate inding Lipid inding Vitamin inding Hromatin inding Metal cluster inding Nucleoside-triphosphatase regulator activity Peroxidase activity Enzyme inhiitor activity Caroxylic acid inding Direct GO Counts of Biological Processes Numer of Sequence Direct GO Counts of Molecular Functions Numer of Sequences

15 Cellular Component c Direct GO Counts of Cellular Components Intracellular organelle Cytoplasm Cytoplasmic part Plasma memrane Intrinsic to memrane Rionucleoprotein complex Plasma memrane part cell wall Thylakoid Thylakoid part Photosynthetic memrane Organelle inner memrane Uiquitin ligase complex Mitochondrial memrane part Endoplasmic reticulum memrane Respiratory chain Photosystem Extrinsic to memrane Nuclear envelope Proteasome complex Proton-transporting two-sector ATPase complex Outer memrane Memrane coat Organelle outer memrane Cytoplasmic vesicle part Coated memrane Respiratory chain complex I NADH dehydrogenase complex Proton-transporting ATP synthase complex Numer of Sequences Supplement Figure The Direct GO count graphs depicting, a) Biological processes ) Cellular components c) Molecular functions in the IGA transcriptome assemly.

16 a Supplement Figure. The relationship etween numer of Go terms and length of sequences. a) Sanger-EST unigenes ) IGA transcriptome contigs.

17 a Supplement Figure. Distriution of annotation score vs. numer of sequences a) Sanger-EST unigenes ) IGA transcriptome contigs.

18 a Supplement Figure. The relationship etween length of sequence and annotation a) Sanger- EST unigenes ) IGA transcriptome contigs.

19 7 Supplement Figure : A flow chart of steps taken to assemle pepper IGA reads. Super assemly comprises of the comined assemly of Velvet K-mers or CLC workench iterations (within each square ox two super assemlies). The assemly of each super assemly is depicted y different colors to show Mega assemlies (immediately elow each ox). The Mega assemlies were comined to make Meta assemly (navy lue ox marked as reference sequence). 8