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We believe that if after running blast, the global identity between a resulting sequence and our query is at least 30%, we can say that two sequences are homologs. what is the difference between local identity and global identity and how can we calculate them? the following file is the global alignment result of one of my PSI-BLAST hits against query:

Aligned_sequences: 2
# 1: NP_418577.1
# 2: WP_036312822.1
# Matrix: EBLOSUM62
# Gap_penalty: 10.0
# Extend_penalty: 0.5
#
# Length: 264
# Identity:      85/264 (32.2%)
# Similarity:   125/264 (47.3%)
# Gaps:          29/264 (11.0%)
# Score: 376.5
# 
#
#=======================================

NP_418577.1        1 -----------MAEMKNLKIE-------VVRYNPEVDTAPHSAFYEVPYD     32
                                ..|..:..|:       :.|::||||..|....|:|...
WP_036312822.      1 MTIVDSGAPADTQEANDSGIQSYLVTFIIRRFDPEVDAEPRWVDYDVEMY     50

NP_418577.1       33 ATTSLLDALGYIKDNLAPDLSYRWSCRMAICGSCGMMVNNVPKLACKTFL     82
                     .|..:||||..||.::...||:|.||...||||..|.:|...:|||||.:
WP_036312822.     51 PTDRVLDALHRIKWDVDGTLSFRRSCAHGICGSDAMRINGRNRLACKTLI    100

NP_418577.1       83 R--DYTDGMKVEALANFPIERDLVVDMTHFIESLEAIKPYIIGNSRTADQ    130
                     :  |.:..:.|||:...|:|:||:|||..|.||...::|::...|.....
WP_036312822.    101 KDLDISKPIYVEAIKGLPLEKDLIVDMDPFFESFRDVQPFLQPKSAPEPG    150

NP_418577.1      131 GTNIQTPAQMAKYHQFSGCINCGLCYAACPQFGLNPEFIGPAAITLAHRY    180
                     ....|:....|.|...:.||.|..|.::||.|..:.::.|||||..|||:
WP_036312822.    151 KERFQSIKDRAVYDDTTKCILCAACTSSCPVFWTDGQYFGPAAIVNAHRF    200

NP_418577.1      181 NEDSRDHGKKERMAQLNSQNGVWSCTFVGYCSEVCPKHVDPAAAIQQGKV    230
                     ..||||.....|:..||.:.|||.|.....|:|.||:.::...||.:.|.
WP_036312822.    201 IFDSRDDAADVRLDILNDKEGVWRCRTTFNCTEACPRGIEITKAIAEVKQ    250

NP_418577.1      231 ESSKDFLIATLKPR    244
                     ...:.         
WP_036312822.    251 AVLRG---------    255


#---------------------------------------
#---------------------------------------
the following file is the local alignment of that hit against query:
Aligned_sequences: 2
# 1: NP_418577.1
# 2: WP_036312822.1
# Matrix: EBLOSUM62
# Gap_penalty: 10.0
# Extend_penalty: 0.5
#
# Length: 219
# Identity:      83/219 (37.9%)
# Similarity:   119/219 (54.3%)
# Gaps:           2/219 ( 0.9%)
# Score: 390.5
# 
#
#=======================================

NP_418577.1       13 RYNPEVDTAPHSAFYEVPYDATTSLLDALGYIKDNLAPDLSYRWSCRMAI     62
                     |::||||..|....|:|....|..:||||..||.::...||:|.||...|
WP_036312822.     31 RFDPEVDAEPRWVDYDVEMYPTDRVLDALHRIKWDVDGTLSFRRSCAHGI     80

NP_418577.1       63 CGSCGMMVNNVPKLACKTFLR--DYTDGMKVEALANFPIERDLVVDMTHF    110
                     |||..|.:|...:|||||.::  |.:..:.|||:...|:|:||:|||..|
WP_036312822.     81 CGSDAMRINGRNRLACKTLIKDLDISKPIYVEAIKGLPLEKDLIVDMDPF    130

NP_418577.1      111 IESLEAIKPYIIGNSRTADQGTNIQTPAQMAKYHQFSGCINCGLCYAACP    160
                     .||...::|::...|.........|:....|.|...:.||.|..|.::||
WP_036312822.    131 FESFRDVQPFLQPKSAPEPGKERFQSIKDRAVYDDTTKCILCAACTSSCP    180

NP_418577.1      161 QFGLNPEFIGPAAITLAHRYNEDSRDHGKKERMAQLNSQNGVWSCTFVGY    210
                     .|..:.::.|||||..|||:..||||.....|:..||.:.|||.|.....
WP_036312822.    181 VFWTDGQYFGPAAIVNAHRFIFDSRDDAADVRLDILNDKEGVWRCRTTFN    230

NP_418577.1      211 CSEVCPKHVDPAAAIQQGK    229
                     |:|.||:.::...||.:.|
WP_036312822.    231 CTEACPRGIEITKAIAEVK    249


#---------------------------------------
#---------------------------------------
terdon
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Sara
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    BLAST (Basic *Local* Alignment Search Tool) doesn't give you global alignments, so you may need to reconsider what you're doing/asking.

    Furthermore, "homolog" has a very specific definition. Homologs are genes that are both descended from the same ancestral sequence. It doesn't, as many often mistake, mean 'similarity'. 30% sequence identity, does not imply homology by itself. You need more information.

     – Joe Healey Jun 21 '17 at 08:49
  • Take a look here (http://www.ebi.ac.uk/Tools/psa/) if you want to find tools for local and global alignment. – Joe Healey Jun 21 '17 at 08:50
  • no it's not my problem. Maybe I couldn't describe it well. I have run PSI-BLAST for my sequence and I 've got some sequences that I believe they are homologs of my sequence, but I'm not sure if they are really homologs or not and which one of these sequences are real homologs. For making sure about this homology, I must check local and global identity and I had done it. Maybe a part of a sequence with mine, has a good identity, but I can't say these two sequences are homologs. They must have globally good identity – Sara Jun 21 '17 at 09:02
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    Perhaps I'm wrong but "They must have globally good identity" still isn't really enough to say that these sequences are homologs. You need ancestry information for the organisms you're interested in.

    If you really want to know orthology/homology, I'd suggest using a dedicated orthology finding program like OrthAgogue, OrthoMCL, or better yet ROARY if your sequences are bacterial.

     – Joe Healey Jun 21 '17 at 09:04
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    No they don't. It all depends on how closely related the species are. Please [edit] your question and add more information. There is no magix sequence simlarity threshold that defines homology. – terdon Jun 21 '17 at 09:04
  • how much the species should be close? – Sara Jun 21 '17 at 09:13
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    There is no magic formula. For closely related species I would expect a sequence identity of far more than 30% for a true homolog but for distant species, I could also expect far lower. As I told you in my answer to your previous question, it is far more important to look at the conservation of specific residues than to look at the overall sequence similarity value. – terdon Jun 21 '17 at 09:15
  • Please don't post images of text. They just make the question slower to load, they cannot be edited and cannot be searched. Instead, just copy/paste the text directly into your question and use the formatting tools to make it appear like code. Also, the alignment is useless if we don't i) know the species being aligned and ii) know the e-value of the alignment. – terdon Jun 21 '17 at 09:32

2 Answers2

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If you want a purely alignment based approach to begin to infer homology (note I say infer, as we stated in the comments, this isn't sufficient to say 2 sequences are homologs), I would use a Multiple Sequence Alignment (MSA) instead of a pairwise like you've done so far, and include as many similar proteins as you can from BLAST.

The choice of algorithm for the MSA probably won't matter too much, as you will be looking for conserved regions anyway.

Once you have your MSA, you can easily spot conserved domains. If anything is known about this protein structurally, it's quite trivial to work out whether a conserved region is a functional domain (active site, that kind of thing).

With this information, you would be somewhat justified in calling these proteins structural homologs at least if they share domains, but, to reiterate, without doing phylogenetic analyses, you cannot call them true homologs.

Joe Healey
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Defining homology isn't as simple as an identity cutoff, however, you're right that homology seems likely given a certain identity. This is where e-value comes into play. See: https://en.wikipedia.org/wiki/Expected_value

To answer the question, global-global requires that the alignment account for every base in the query and the target sequence. local-global requires a the full length of the query on a part of the target. local-local allows only the highest scoring part of the alignment between the query and target to be reported. i.e. one functional domain against another. these hits are often called HSPs.

Dan
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