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Current Laccaria transcriptomics ressources: cDNA libraries, ESTs, cDNA arrays

Current Laccaria transcriptomics ressources: cDNA libraries, ESTs, cDNA arrays. A Kohler, M Peter, F Duchaussoy, A Deveau, C Delaruelle P Brokstein, E Lindquist, PM Richardson et F Martin. I. cDNA libraries. CAHB, CAHC, CAHF,CAHG, cDNA libraries from mixed tissues (JGI)

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Current Laccaria transcriptomics ressources: cDNA libraries, ESTs, cDNA arrays

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  1. Current Laccaria transcriptomics ressources: cDNA libraries, ESTs, cDNA arrays A Kohler, M Peter, F Duchaussoy, A Deveau, C Delaruelle P Brokstein, E Lindquist, PM Richardson et F Martin

  2. I. cDNA libraries CAHB, CAHC, CAHF,CAHG, cDNA libraries from mixed tissues (JGI) CAHH, CAPI,CAPN (mainly mycelium) 30826 ESTs LU ? 165 ESTs Lb01-Lb34 cDNA libraries from three week old mycelium (INRA Nancy) 1636 ESTs LbII cDNA library from fruiting bodies stage 1&2 (INRA Nancy) 496 ESTs LbIII cDNA library from cap of fruiting body stage 6 (INRA Nancy) 469 ESTs LG0ADF cDNA library from mycorrhizal roots (Douglas Fir) (INRA Nancy& Génoscope) 747 ESTs LG0ADG cDNA library from mycorrhizal roots (Poplar) (INRA Nancy& Génoscope) 1025 ESTs total 4373 ESTs

  3. II. ESTs 14312 Tentative consensus sequences (TCs) 11697 Blast Vs Laccaria Gene Models 146 Blast Vs Mitochondrial Genome 33 Blast Vs Poplar 1245 Blast Vs Laccaria Genome 24 Blast Vs Laccaria Non-assembled Genome 181 Unknown 986 Bad Sequences 14312 seqs Blast Vs Laccaria Gene Models 11697 seqs Blast Vs Laccaria Genome 1245 seqs Blast Vs Mitochondrial Genome 146 seqs Blast Vs Poplar Genome 33 seqs Blast Vs Laccaria Non-Assembled Sequences 24 seqs Select By Quality of Sequences E-value limit 1e-20 181 good seqs 986 bad seqs F. Duchaussoy

  4. Functional classification of TCs using BlastX and KOG 50% unknown F. Duchaussoy EuKaryotic Orthologous Groups (KOG)

  5. F. Duchaussoy

  6. Most abundant transcripts 31 TCs with more than 50 ESTs 410 TCs with contain between 10 and 50 ESTs 10695 TCs with one or two ESTs

  7. Major TC: 585 ESTs (14 ESTs Lb, 2 ESTs LG0ADG ,569 ESTs mixed libraries) ???

  8. Most abundant transcripts Contig ESTs gene model putative function 1323708:239 585 eu2.Lbscf0029g00760 Transcription initiation factor TFIID, subunit BDF1 and related bromodomain proteins 1323711:21 348 estExt_fgenesh2_pg.C_60136 Ribosomal protein S12/S23 1323711:20 281 estExt_fgenesh2_pg.C_60136 Ribosomal protein S12/S23 1323708:238 242 fgenesh3_pg.C_scaffold_6000144 - 1323708:237 206 estExt_fgenesh2_pg.C_250083 Nucleolar GTPase/ATPase p130 1323708:236 199 estExt_fgenesh2_pg.C_250083 Nucleolar GTPase/ATPase p130 1316099:2 157 no hit - 1323708:232 136 fgenesh3_pg.C_scaffold_10000205 jgi|Phchr1|7088|fgenesh1_pg.C_ scaffold_12000272 1323708:233 133 fgenesh3_pg.C_scaffold_10000205 jgi|Phchr1|7088|fgenesh1_pg.C_ scaffold_12000272 1323708:235 133 eu2.Lbscf0068g00130 jgi|Phchr1|7088|fgenesh1_pg.C_ scaffold_12000272

  9. 20 614 gene models 14312 (11697) TCs 6514 gene models with EST support -Transcripts from the same gene model in different TCs (bad quality,allelic, assembling errors?) -no overlapp between ESTs from 5’ and 3’ end of the gene -Several ESTs from one gene due to altered splicing? Only one-third of the predicted gene models have an EST support so far!

  10. 199 ESTs 206 ESTs Example: gene model with multiple TC support

  11. TC sequence don’t fit with the manually annotated gene model Example 1: annotaded by A.Deveau: Methyl malonate semi aldehyde dehydrogenase

  12. TC sequence don’t fit with the manually annotated gene model Example 2: 1 EST in TC Not sufficient? Annotated by M. Reich: Acyl-CoA thioester hydrolase

  13. Example: Altered splicing of the transcript Annotated by M. Reich: NADH-cytochrome b5 reductase

  14. Existing nylon arrays Planned microarrays PICME ARC Seibersdorf research GmbH Custom tailored DNA Chips 1636mycelium cDNA library 965 fruiting body cDNA library 1772 mycorrhizal cDNA libraries (747 Douglas Fir/1025 Poplar) 4373 ESTs Laccaria nylon array I: 4608 ESTs spotted (mycelium cDNA library 951 ESTs, each spotted four times) ~150-200 Euros Whole genome oligochip Nimblegene Operon? Laccaria nylon array II: 4992 ESTs spotted (mycelium cDNA library 768 ESTs, fruiting body cDNA libraries 4224 clones (965 sequenced)) Costs? III. Microarrays

  15. Principal component analysis of gene expression in Laccaria under various conditions Microarray experiments by M. Peter

  16. Example: A Laccaria transcript regulated upon contact with certain bacteria without corresponding gene model Microarray experiment by A. Deveau

  17. Example: Laccaria-specific transcripts/gene models Microarray experiment by A. Deveau

  18. DISCUSSION Planned microarrays PICME ARC Seibersdorf research GmbH Custom tailored DNA Chips 1636mycelium cDNA library 965 fruiting body cDNA library 1772 mycorrhizal cDNA libraries (747 Douglas Fir/1025 Poplar) 4373 ESTs -more ESTs needed? libraries from various tissues to get more information about transcribed genes? (developmental stages, various conditions?) -Construction of microarrays: DNA Chips and/or Oligo Chips? ~150-200 Euros Whole genome oligochip Nimblegene? Operon? Costs?

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