#@ 16122019 Dear Vasilis, I have added the fastq files you mention to the directory, sorry for the omission. Concerning the number of reads, there is no 1:1 relation between fastq and bam since a read might not be mapped, or a read might be multi-mapped. Concerning the name " Dr-Stath" in the filenames, please ignore this (and opt. rename the files), as some of the runs contained also other samples, the name of the run always contains all contributors. BW #@ Pseudomonas RNA-seq data Vasilis Promponas Sun, Dec 15, 2019 at 12:30 AM To: Martin Reczko Cc: Pantelis Hatzis , Giorgos E Apidianakis Dear Martin, We have gone through all the fastq files you have sent us and it seems that files originating from - run347 (YAbR21-OD3-B133-3, YAbR24-OD3-C3719-3) - run354 (YAbR34r-OD3-PACS2-1, YAbR36-OD3-PACS2-3, YAbR4-OD1-C3719-1, YAbR5r-OD1-C3719-2, YAbR25- OD3-CF5-1, YAbR27-OD3-CF5-3) - run360 (YAbR3-OD1-B133-3) # not sure whether this file was used for mapping reads are not available under http://genomics-lab.fleming.gr/fleming/external/Apidianakis/fastq/ I believe we will need the above for submission as well. Another (perhaps naive question): shouldn't the total numbers of reads in fastq files per biological condition (mentioned in the bottom of http://genomics-lab.fleming.gr/fleming/external/Apidianakis/all-samples1.txt) match with the mapped reads in the .bam files? I computed counted reads using samtools under bash for i in *.bam; do echo $i; ~/samtools-1.10/samtools view -c $i; done and they match with the subread/featurecounts statistics available with the bam files. In fact, the total reads in the fastq files seem to be systematically less (except: YAbR21-OD3-B133-3, YAbR24-OD3-C3719-3); in most cases read number difference is approximately two-fold (see attached text file). Am I missing something here? Kind regards, Vasilis -------------------------------------------------- Vasilis Promponas, PhD Assistant Professor in Bioinformatics Head, Bioinformatics Research Laboratory Department of Biological Sciences New Campus, Office B161 University of Cyprus P.O. Box 20537 CY 1678, NICOSIA CYPRUS tel: 00357-22892879 (office) 00357-22893972 (lab) fax: 00357-22895095 (departmental) email: vprobon@ucy.ac.cy, vprobon@biol.uoa.gr, vasilis.promponas@gmail.com web: http://troodos.biol.ucy.ac.cy On Wed, Dec 11, 2019 at 7:22 PM Martin Reczko wrote: Dear Vasilis, at http://genomics-lab.fleming.gr/fleming/external/Apidianakis/fastq/ I have collected all fastq files. Information about these and how some were merged is in http://genomics-lab.fleming.gr/fleming/external/Apidianakis/all-samples1.txt BW, Martin #@ Hi all, I see the OD3 data. What about OD1? So the new lists are essentially the same? Regards, Yiorgos **************************************************************************** Yiorgos Apidianakis, Ph.D., Assistant Professor 1 Panepistimiou Ave., Department of Biological Sciences University of Cyprus, 2109 Aglatzia, Cyprus Phone: +357-22893767 (office) or +357-22893968 (lab) e-mail: apidiana@ucy.ac.cy P.O.Box 20537, 1678 Nicosia, Cyprus http://ucy.ac.cy/dir/en/component/comprofiler/userprofile/apidiana #@ Tuesday, February 13, 2018 3:51:23 PM Dear all, at http://genomics-lab.fleming.gr/fleming/external/Apidianakis/ribominus are the Deseq2 results after exclusion of the 76 (ribosomal) genes defined by Vasilis. The following entry did not occur in our previous result (all.csv): NC_008463.1 RefSeq sequence_feature 759485 759542 . + . ID=id13 Note=23S ribosomal RNA rRNA prediction is too short gbkey=misc_feature Best wishes, Martin Fwd: Pseudomonas RNAseq: non protein coding transcripts for depletion Vasilis Promponas Fri, Feb 9, 2018 at 2:18 PM To: Martin Reczko Cc: Pantelis Hatzis We have actually used an orthogonal approach to get these genes and verified with the gene_biotype information. Could you possibly re-run the first parts of the analysis (normalization & differential expression analysis) after excluding these genes or we have to set up the pipeline on our servers?? All the best, Vasilis -------------------------------------------------- Vasilis Promponas, PhD Assistant Professor in Bioinformatics Head, Bioinformatics Research Laboratory Department of Biological Sciences New Campus, Office B161 University of Cyprus P.O. Box 20537 CY 1678, NICOSIA CYPRUS tel: 00357-22892879 (office) 00357-22893972 (lab) fax: 00357-22895095 (departmental) email: vprobon@ucy.ac.cy, vprobon@biol.uoa.gr, vasilis.promponas@gmail.com web: http://troodos.biol.ucy.ac.cy #@ Liao Y, Smyth GK and Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics, 30(7):923-30, 2014 Dear Yiorgos, the requested comparisons are at: http://genomics-lab.fleming.gr/fleming/external/Apidianakis/run362/groups2 The files below contain genes that are diff. expressed with a corrected P value of <0.05 : deseq2_PA14_OD1_vs_PACS2_OD1-sig.csv deseq2_PA14_OD1_vs_CF5_OD1-sig.csv deseq2_PA14_OD1_vs_C3719_OD1-sig.csv deseq2_MTB_OD1_vs_PACS2_OD1-sig.csv deseq2_MTB_OD1_vs_CF5_OD1-sig.csv deseq2_MTB_OD1_vs_C3719_OD1-sig.csv deseq2_B133_OD1_vs_PACS2_OD1-sig.csv deseq2_B133_OD1_vs_CF5_OD1-sig.csv deseq2_B133_OD1_vs_C3719_OD1-sig.csv deseq2_PA14_OD3_vs_PACS2_OD3-sig.csv deseq2_PA14_OD3_vs_CF5_OD3-sig.csv deseq2_PA14_OD3_vs_C3719_OD3-sig.csv deseq2_MTB_OD3_vs_PACS2_OD3-sig.csv deseq2_MTB_OD3_vs_CF5_OD3-sig.csv deseq2_MTB_OD3_vs_C3719_OD3-sig.csv deseq2_B133_OD3_vs_PACS2_OD3-sig.csv deseq2_B133_OD3_vs_CF5_OD3-sig.csv deseq2_B133_OD3_vs_C3719_OD3-sig.csv Concatenating these give the consensus lists you mention. The same files without "-sig" in their name contain all results. BW, Martin #@ #@ On Wed, Sep 13, 2017 at 12:49 PM, Yiorgos Apidianakis wrote: Hi Pantelis, while the computational part of our work is pending, we would appreciate if Martin could produce the following gene expression comparison xls lists, plus 2 consensus lists one per each OD: 1. PA14 OD1, vs CF5 OD1 16 2. PA14 OD1, vs C3719 OD1 3. PA14 OD1, vs PACS2 OD1 4. MTB-1 OD1, vs CF5 OD1 5. MTB-1 OD1, vs C3719 OD1 6. MTB-1 OD1, vs PACS2 OD1 7. B136-33 OD1 vs CF5 OD1 8. B136-33 OD1vs C3719 OD1 9. B136-33 OD1 vs PACS2 OD1 The consensus list of 1-9 gene lists (each containing only the genes that are diff. expressed with a corrected P value of <0.05) 10. PA14 OD3, vs CF5 OD3 11. PA14 OD3, vs C3719 OD3 12. PA14 OD3, vs PACS2 OD3 13. MTB-1 OD3, vs CF5 OD3 14. MTB-1 OD3, vs C3719 OD3 15. MTB-1 OD3, vs PACS2 OD3 16. B136-33 OD3 vs CF5 OD3 17. B136-33 OD3 vs C3719 OD3 18. B136-33 OD3 vs PACS2 OD3 The consensus list of 10-18 gene lists (each containing only the genes that are diff. expressed with a corrected P value of <0.05) Thanks Yiorgos **************************************************************************** Yiorgos Apidianakis, Ph.D., Assistant Professor 1 Panepistimiou Ave., Department of Biological Sciences University of Cyprus, 2109 Aglatzia, Cyprus Phone: +357-22893767 (office) or +357-22893968 (lab) e-mail: apidiana@ucy.ac.cy P.O.Box 20537, 1678 Nicosia, Cyprus http://ucy.ac.cy/dir/en/component/comprofiler/userprofile/apidiana #@ apidianakis.giorgos@ucy.ac.cy vasilis.promponas@gmail.com Dear Dr Apidianakis, the analysis of your samples has finished. To facilitate differential expression analysis, all samples have been aligned to UCBPP-PA14 as a common reference. The UCBPP-PA14 assembly has the ID GCF_000014625.1_ASM1462v1_genomic.fna and the annotation GCF_000014625.1_ASM1462v1_genomic.gff. The bam files with reads and alignments are in the directory: http://genomics-lab.fleming.gr/fleming/external/Apidianakis/run362/bam-files The files and the number of reads are: YAbR10-OD1-MTB-1.bam.sorted.bam 12057732 YAbR11-OD1-MTB-2.bam.sorted.bam 9572335 YAbR13-OD1-PA14-.bam.sorted.bam 7477877 YAbR14-OD1-PA14-.bam.sorted.bam 7223463 YAbR17-OD1-PACS2-2.bam.sorted.bam 10785889 YAbR18-OD1-PACS2-3.bam.sorted.bam 12216375 YAbR1-OD1-B133-1.bam.sorted.bam 7613469 YAbR20r-OD3-B133-2.bam.sorted.bam 12331355 YAbR21-OD3-B133-3.bam.sorted.bam 8687847 YAbR22-OD3-C3719-1.bam.sorted.bam 14515133 YAbR24-OD3-C3719-3.bam.sorted.bam 8868728 YAbR25-OD3-CF5-1.bam.sorted.bam 9708937 YAbR27-OD3-CF5-.bam.sorted.bam 14507695 YAbR28r-OD3-MTB-1.bam.sorted.bam 23942790 YAbR30-OD3-MTB-3.bam.sorted.bam 18617006 YAbR32-OD3-PA14-2.bam.sorted.bam 16048660 YAbR33-OD3-PA14-3.bam.sorted.bam 14727806 YAbR34r-OD3-PACS2-.bam.sorted.bam 11968488 YAbR36-OD3-PACS2-3.bam.sorted.bam 10791272 YAbR3-OD1-B133-3.bam.sorted.bam 12793627 YAbR4-OD1-C3719-1.bam.sorted.bam 11968488 YAbR5r-OD1-C3719-.bam.sorted.bam 20572548 YAbR8-OD1-CF5-2.bam.sorted.bam 18704462 YAbR9-OD1-CF5-3.bam.sorted.bam 12033907 If you load the UCBPP-PA14 assembly into IGV, you can visualize all reads aligments (download also the corresponding *.bai file). For diff. exp. analysis, the groups PA14, MTB-1, B136-33 vs CF5, C3719 and PACS2 are named "pa14","mtb1","b136","cf5","c3719","pacs2". Using Deseq2, the number of diff. exp. genes with an adjusted p-value below 0.05 are: contrast n_genes_with_padj_lt_0.05 pa14_vs_mtb1 571 pa14_vs_b136 407 pa14_vs_cf5 1914 pa14_vs_c3719 1148 pa14_vs_pacs2 1092 mtb1_vs_b136 157 mtb1_vs_cf5 1149 mtb1_vs_c3719 633 mtb1_vs_pacs2 755 b136_vs_cf5 1145 b136_vs_c3719 461 b136_vs_pacs2 675 cf5_vs_c3719 753 cf5_vs_pacs2 1131 c3719_vs_pacs2 829 The results for each contrast are in the file: deseq2_[contrast].xlsx and an MAplot has been generated in: MAplot_[contrast].png I have included the UCBPP-PA14 sequence and annotation as well as the analysis script (diff-exp-deseq2.r) also in the directory. Let me know if you need further information. Kind regards, Martin -- Dr. Martin Reczko Head of Genomic Bioinformatics Functional Genomics Laboratory and Genomics Facility Institute of Molecular Biology and Genetics Biomedical Sciences Research Center "Alexander Fleming" iTechnical Coordinator - ELIXIR Greece 34 Fleming Street 16672, Vari, Greece Tel. +30-210-9656310 FAX: +30-210-9653934 email: reczko@fleming.gr ---------- Forwarded message ---------- From: Yiorgos Apidianakis Date: 11 July 2017 at 15:13 Subject: Strain comparisson To: Pantelis Hatzis Panteli, regarding bacteria transcriptomics we need to pinpoint differences in gene expression between: PA14, MTB-1, B136-33 vs CF5, C3719 and PACS2 Yiorgos