1. 1 Discussion Practical 1

2. Features of major databases (PubMed and NCBI Protein Db) 2

3. Anatomy of PubMed Db 3

4. Epub ahead of print and journal impact factor 4 How to get impact factor of any journal: 1) Direct source – web of science database 2) In direct source, e.g. blogs, sites etc (do Google search) Adopted from : http://admin-apps.isiknowledge.com/JCR/JCR?RQ=LIST_SUMMARY_JOURNAL

5. Anatomy of a PubMed record 5

6. Demo on downloading articles 6

7. Anatomy of a Protein Db 7

8. 8 Other popular sources: dbj – DDBJ (DNA Data Bank of Japan database) emb – The European Molecular Biology Laboratory (EMBL) database prf – Protein Research Foundation database sp – SwissProt gb – GenBank pir – Protein Information Resource Version NM_000546.3 GI or Geninfo Identifier) 120407067 Source Refseq database Accession NM_000546 Accession numbers and GenInfo Identifiers

9. 9 Why do we need accession number and GI for one record? 1) What is the difference between accession and GI? 2) Why do we need these two when both seem to be accession numbers?

10. 10 Q1) Which revision will NCBI show if you were to search by the accession only without the version number? Sequence_v1 NM_000546 Sequence_v2 NM_000546 Sequence_v3 NM_000546 NM_000546.1 NM_000546.2 NM_000546.3 4507636 8400737 120407067 Sequence update Sequence update GI Version Why do we need accession number and GI for one record?

11. 11 Accession numbers -The unique identifier for a sequence record. -An accession number applies to the complete record. -Accession numbers do not change, even if information in the record is changed at the author's request. -Sometimes, however, an original accession number might become secondary to a newer accession number, if the authors make a new submission that combines previous sequences, or if for some reason a new submission supercedes an earlier record.

12. 12 GenInfo Identifiers - GenInfo Identifier: sequence identification number - If a sequence changes in any way, a new GI number will be assigned - A separate GI number is also assigned to each protein translation Within a nucleotide sequence record -A new GI is assigned if the protein translation changes in any way -GI sequence identifiers run parallel to the new accession.version system of sequence identifiers

13. 13 Version -A nucleotide sequence identification number that represents a single, specific sequence in the GenBank database. -If there is any change to the sequence data (even a single base), the version number will be increased, e.g., U12345.1 → U12345.2, but the accession portion will remain stable. -The accession.version system of sequence identifiers runs parallel to the GI number system, i.e., when any change is made to a sequence, it receives a new GI number AND an increase to its version number. -A Sequence Revision History tool (http://www.ncbi.nlm.nih.gov/entrez/sutils/girevhist.cgi) is available to track the various GI numbers, version numbers, and update dates for sequences that appeared in a specific GenBank record

14. 14 Anatomy of a Protein Db record

15. 15 Fasta Sequence

16. Fasta Format Text-based format for representing  nucleic acid sequences or peptide sequences (single letter codes). Easy to manipulate and parse sequences to programs. >SEQUENCE_1 MTEITAAMVKELRESTGAGMMDCKNALSETNGDFDKAVQLLREKGLGKAAKKADRLAAEG LVSVKVSDDFTIAAMRPSYLSYEDLDMTFVENEYKALVAELEKENEERRRLKDPNKPEHK IPQFASRKQLSDAILKEAEEKIKEELKAQGKPEKIWDNIIPGKMNSFIADNSQLDSKLTL MGQFYVMDDKKTVEQVIAEKEKEFGGKIKIVEFICFEVGEGLEKKTEDFAAEVAAQL >SEQUENCE_2 SATVSEINSETDFVAKNDQFIALTKDTTAHIQSNSLQSVEELHSSTINGVKFEEYLKSQI ATIGENLVVRRFATLKAGANGVVNGYIHTNGRVGVVIAAACDSAEVASKSRDLLRQICMH Description line/row Sequence data line(s) Description line/row Sequence data line(s)

17. Fasta Format (cont.) Begins with a single-line description, followed by lines of sequence data. Description line –Distinguished from the sequence data by a greater-than (">") symbol. –The word following the ">" symbol in the same row is the identifier of the sequence. –There should be no space between the ">" and the first letter of the identifier. –Keep the identifier short and clear ; Some old programs only accept identifiers of only 10 characters. For example: > gi|5524211|Human or >HumanP53 Sequence line(s) –Ensure that the sequence data starts in the row following the description row (be careful of word wrap feature) –The sequence ends if another line starting with a ">" appears; this indicates the start of another sequence. >SEQUENCE_1 MTEITAAMVKELRESTGAGMMDCKNALSETNGDFDKAVQLLREKGLGKAAKKADRLAAEG LVSVKVSDDFTIAAMRPSYLSYEDLDMTFVENEYKALVAELEKENEERRRLKDPNKPEHK IPQFASRKQLSDAILKEAEEKIKEELKAQGKPEKIWDNIIPGKMNSFIADNSQLDSKLTL MGQFYVMDDKKTVEQVIAEKEKEFGGKIKIVEFICFEVGEGLEKKTEDFAAEVAAQL >SEQUENCE_2 SATVSEINSETDFVAKNDQFIALTKDTTAHIQSNSLQSVEELHSSTINGVKFEEYLKSQI ATIGENLVVRRFATLKAGANGVVNGYIHTNGRVGVVIAAACDSAEVASKSRDLLRQICMH Description line/row Sequence data line(s) Description line/row Sequence data line(s)

18. Amino acids & Nucleotides 18

20. IUPAC One Letter Amino Acid Code A B C D E F G H I J K L M N O P Q R S T U V W X Y Z Alanine Cysteine Glycine Histidine Isoleucine Leucine Methionine Proline Serine Threonine Valine Glutamic Acid Aspartic Acid Phenylalanine Lysine Asparagine Glutamine Arginine Tryptophan Tyrosine 21 st (Sec) Selenocysteine 22 nd (Pyl) Pyrrolysine GLx ASx Glutamic Acid Aspar(D)ic Acid (F)enylalanine Lysine Asparagi(N)e (Q)lutamine (R)ginine T(W)ptophan T(Y)rosine 21 st (Sec)Selenocysteine 22 nd (Pyl) Pyrr(O)lysine GLx ASx

21. Note Amino acidThree letter codeSingle letter code Asparagine or aspartic acidAsxB Glutamine or glutamic acid,GLxZ Leucine or Isoleucine,XleJ Unspecified or unknown amino acidXaaX

22. 22 Standard IUPAC Nucleotide code is used to describe ambiguous sites in a given DNA sequence motif, where a single character may represent more than one nucleotide. The code is shown in the table below. IUPAC Nucleotide Code http://www.yeastract.com/help/help_iupac.php

23. Advice We highly recommend that you memorize the amino acid codes and their structures Memorizing the codes and in particular the structures will be very useful for this module and other modules, especially for research purposes. It is not compulsory that you memorize these for this module.

24. Features of major database (Gene Db) 24

25. 25 Anatomy of Gene Db

26. 26 Anatomy of a Gene Db record

27. A section of Gene Db record: Reference Sequences 27 mRNA Accession number Protein Accession number

28. 28 Nucleic Acid Databases Entrez nucleotide database (nt) GenBank DDBJ EMBL RefSeq_genomic

29. 29 Amino Acid Databases 1) Sequence repositories GenPept (redundant; translation of GenBank; minimal annotation) Entrez Protein (redundant or NR) translated DDBJ/EMBL/GenBank ( i.e. GenPept) Swiss-Prot, PIR, RefSeq_protein and PDB RefSeq (non-redundant; reference sequences; minimal manual curation; limited species) 2) Universal curated databases PIR-PSD (non-redundant; focus on protein family classification) Swiss-Prot (non-redundant; manually annotated) TrEMBL (non-redundant; extensively computer-annotated) 3) Next-generation of protein sequence database UniProtKB (Swiss-Prot, TrEMBL and PIR-PSD integrated; less redundant than UniProt NREF) UniParc (like Entrez Protein but more comprehensive) UniProt NREF (like RefSeq but more comprehensive and rich with annotation) Read more: http://www.ebi.ac.uk/panda/pdf/apweiler_bairoch_2004.pdfhttp://www.ebi.ac.uk/panda/pdf/apweiler_bairoch_2004.pdf

30. 30 The RefSeq Project Goal: a “ comprehensive, integrated, non-redundant set of sequences, including genomic DNA, transcript (RNA), and protein products, for major research organisms. ” http://www.ncbi.nlm.nih.gov/RefSeq/index.html Designed to reduce duplication by selecting one representative sequence for each locus, except when there are naturally occurring paralogs and splice variants. Info from: –Predictions from genomic sequence –Analysis of GenBank Records –Collaborating databases

31. Genbank versus refseq http://www.ncbi.nlm.nih.gov/books/NBK21105/#ch1.Appendix_GenBank_RefSeq_TPA_and_UniP

32. Choice of databases for genomic/proteomic data Promoter Enhancer Gene EE I UU Nucleotide Protein RefSeq_genomeRefSeq_Protein Gene All of above in multiple records All real/ reliably predicted proteins in multiple records Reference ones only Reference proteins only Gene record with all related Information included (mRNA Protein, promoter, enhancer) Genome architecture Databases to house genomic/proteomic data

33. Database searching can help answer questions like What is the sequence of human IL-10? What is the gene coding for human IL-10? Is the function of human IL-10 known? What is it? Are there any variants of human IL-10? Who sequenced this gene? What are the differences between IL-10 in human and in other species? Which species are known to have IL-10? Is the structure of IL-10 known? What are structural and functional domains of the IL-10? Are there any motifs in the sequence that explain their properties? What is an upstream region of IL-10 containing transcriptional regulation sites? IL10 = X?

34. Take home messages for databases Bioinformatics = databases + tools General databases versus specialized databases Databases come and go (especially the small ones) Database redundancy - many databases for the same topic (use the most comprehensive, if not use all for comprehensiveness) Database accuracy – published ones are more reliable; nevertheless, they are still prone to errors; always good to spend sometime assessing the reliability of your data of interest by doing cross-referencing to literature or other databases Fortunately, most databases are cross-referenced Unfortunately, no common standard format; need to spend some time familiarizing each; becomes easy after some practice Finding databases relevant to you –NAR Database catalogue –Pubmed –Google 2 main methods for searching databases (each with its own pros and cons) –1. Keyword search (covered today) –2. Sequence search (day 2) 34