NCBI Blast, Sequence Analysis & Result Interpretation: Lecture 2 part 2 by Dr. Muhammad Naveed

NCBI Blast, Sequence Analysis, Result Interpretation & Result Submission.
Background
BLAST, which The New York Times called the Google of biological research,[2] is one of the most widely used bioinformatics programs for sequence searching.[3] It addresses a fundamental problem in bioinformatics research. The heuristic algorithm it uses is much faster than other approaches, such as calculating an optimal alignment. This emphasis on speed is vital to making the algorithm practical on the huge genome databases currently available, although subsequent algorithms can be even faster.
Input
Input sequences (in FASTA or Genbank format) and weight matrix.
Output
BLAST output can be delivered in a variety of formats. These formats include HTML, plain text, and XML formatting. For NCBI's web-page, the default format for output is HTML. When performing a BLAST on NCBI, the results are given in a graphical format showing the hits found, a table showing sequence identifiers for the hits with scoring related data, as well as alignments for the sequence of interest and the hits received with corresponding BLAST scores for these. The easiest to read and most informative of these is probably the table.
If one is attempting to search for a proprietary sequence or simply one that is unavailable in databases available to the general public through sources such as NCBI, there is a BLAST program available for download to any computer, at no cost. This can be found at BLAST+ executables. There are also commercial programs available for purchase. Databases can be found from the NCBI site, as well as from Index of BLAST databases (FTP).
Process
Using a heuristic method, BLAST finds similar sequences, by locating short matches between the two sequences. This process of finding similar sequences is called seeding. It is after this first match that BLAST begins to make local alignments. While attempting to find similarity in sequences, sets of common letters, known as words, are very important. For example, suppose that the sequence contains the following stretch of letters, GLKFA. If a BLAST was being conducted under normal conditions, the word size would be 3 letters. In this case, using the given stretch of letters, the searched words would be GLK, LKF, KFA. The heuristic algorithm of BLAST locates all common three-letter words between the sequence of interest and the hit sequence or sequences from the database. This result will then be used to build an alignment. After making words for the sequence of interest, the rest of the words are also assembled. These words must satisfy a requirement of having a score of at least the threshold T, when compared by using a scoring matrix.
Algorithm
To run the software, BLAST requires a query sequence to search for, and a sequence to search against (also called the target sequence) or a sequence database containing multiple such sequences. BLAST will find sub-sequences in the database which are similar to sub sequences in the query. In typical usage, the query sequence is much smaller than the database, e.g., the query may be one thousand nucleotides while the database is several billion nucleotides.
#NCBIBlast #BLAST #QueryCoverage

[@bioworld7565]

you are a talent of Pakistan

[@mariachaudhary4854]

Lagta tha ab yeh lecture dobra sun' ne ko nahi mile gaa but sir you did it😇😇

[@Prof.Dr.MuhammadNaveed]

pleasures

[@muhammadyaqoob8843]

Great sir Really you made much difficult task easy for students Appreciate your efforts

[@nimramushtaq4508]

It's like a blessing in disguise Sir.I'm a student of BS(Hons.) Zoology Department, actually i was searching BLAST for my presentation ..i did not found it in a precise way..finally a day before my presentation ,i found your lecture. i hope it would help me tomorrow ..Thank you very much..you did a fantastic job..keep it up Sir...

[@Prof.Dr.MuhammadNaveed]

kzbin.info/www/bejne/noSmpmScrcRnl6M

[@diverseujjwal2619]

Really informative and great lectures. Thanks a lot sir.

[@Prof.Dr.MuhammadNaveed]

You are most welcome

[@asifakram7060]

excellent

[@jigyasatiwari]

Great sir, This video is so helpful for me....can you make the video on " interpretation of the clustal omega results (alignments, result summary, phylogenetic tree [ real ]. I need of this video till Sunday 🥺...please can you make sir. I will be very thankful to you 🙏

[@professortraveler]

Your lectures are awesome Sir, Your lectures are very easy to understand. Keep up the good work.

[@Prof.Dr.MuhammadNaveed]

Thank you, I will

[@MujahidAli-dz6ln]

Really Good Work! Please keep it up.

[@Prof.Dr.MuhammadNaveed]

welcome and sure dear

[@zeekhan4040]

I love your videos. Excellent job.

[@babarbch]

Informative lecture Excelent

[@Prof.Dr.MuhammadNaveed]

welcome dear

[@kazmi_collection7065]

@@Prof.Dr.MuhammadNaveed sir u r amazing

[@A4arooj]

There is one question, that keep on pinching me; can't query coverage be defined as a factor which describes the homology or evolutionary relationship between two sequences. as you mentioned in the class that it always must be 100.... which according to my understanding actually tells weather or not they are homologous to each other.

[@atif8183]

Thank you so much sir! Love from Gomal university D.i Khan. aap nay both easy tarekay say samjha diya mashallah ❤️

[@Prof.Dr.MuhammadNaveed]

pleasures dear

[@bukhtaweranwer1352]

Brilliant

[@jazzypakistan140]

I used to watch your videos very often .. and maine aaj ghor kia k aap ko mai jaanti hu mili b hu.🤣🤣🤣

[@aqsakashigar2282]

Thanks sir ❤️

[@sanianisar6184]

Assalamu Alikum Sir, I am a phd Student and now doing work on transcriptome analysis in garlic.how i will interpret my result using bioinformatices.kindly let me know step wise and dataset which will be good for this species.? Regards

[@uzmaramzan4654]

Great sir. Your lectures are very helpful.

[@Prof.Dr.MuhammadNaveed]

Glad to hear that

[@Mr.Abd0001]

Aslamulikum sir. Sir apka bioinformatics wala playlist sequence main nahi. Samjh nai ati kon si video sy start laen. Kindly guide.

[@swatikanani323]

Whole genome sequencing results interpretation

[@Prof.Dr.MuhammadNaveed]

sure very soon

[@badirajpoot3740]

assalam o alaikum dear sir, sir ye Blast ma description ki thori c guidelines dobara dijiya ga kch smaj ni ah rhi. sir for example hamara sequence 1584 bp ka ha lakin uski first description ma 2918 show ho rha ha max score or total score , agay query cov and persent identity ka boht difference ha ...

[@Prof.Dr.MuhammadNaveed]

waslam just follow identities and coverage not max score

[@mamtapanda6567]

Which one is more significant when comparing sequences for its function prediction....E value or identity % and why How much E value can be considered significant?

[@Prof.Dr.MuhammadNaveed]

both and E value should be 0 or less than 0

[@ridwanunuhuahmad5945]

Hello Sir, I need more clarification on how to run blast and phylogenetic analysis. I have my sequence result from 4 18S rRNA isolate. I run blastn and all the 4 isolate have 95+ percent identity with previously published sequence/ species in the NCBI which is C.parvum. other published species showed lower than 95+ percent identity and when I run phylogenetic tree some of the isolate attached to Cryptosporidium tortoise instead of C. parvum that showed higher percentage identity. How do I interprete phylogenetic tree ? What do I do to identity species from my sequence result and do I deposit my sequence result to NCBI to obtain my sequence assertion number ? Thank you

[@havvaunal3957]

hi, when I run the global align, I get the different identities percentage for the same sequences each time. Why, can you explain this? thank you

[@waqarhussain4406]

Thanks you too much 💝

[@experimentaleducation-by1cy]

in case of new strains similarity would be less than 85%? and can we claim based on these result that' it's new starins ..( I am referring fungal starin here ) or we need to go further gene analysis ,please guide about it if you can ?thanks

[@sultanajannat15]

How to find out the function of an unknown gene using BLAST?

[@adilqadir3407]

Sir do you have any reference which we follow and we can write our blast like that...??

[@yasirnawaz3910]

Sir kuch topic send kru wo lecture prepare kroge

[@farooqhaider7365]

Dear sir, I hope u will be fine. Sir which test is further proceed for novel genus of organism. Plz explain

[@imtiazahmad9238]

use of query and subject ? how to select range or how it varies?

[@imadkhan2029]

Sir can you upload lecture on Illumina Sequencing and Amplicon sequencing .

[@sahajlaboratory7342]

NICE LECTURE ,i, m a research student , few quereies like i hv result of my query seq from ncbi blast which shows 86.88% identity ,so is it ok. further i hv wgs results of a bacteria,in which i wanna go for pathogeny island prediction for which i was using PAIDB v2.0.cn u pls help me how to use tht particular software??

[@Prof.Dr.MuhammadNaveed]

will make video on it

[@jojithomas587]

VERY INFORMATIVE VIDEO. Sir, i have the sequence result but when i use blast the similarity is different in term of organism name as well as the gene. is that possible? kindly advice. thank you.

[@Prof.Dr.MuhammadNaveed]

yes but for only for some conserved genes like 16S rRNA

[@iqrasaeed642]

sir what if I get the negative E-value? should I have to select only 0.0 e-values for comparison?

[@Prof.Dr.MuhammadNaveed]

yes but query coverage and percentage identity as well

[@mohdawas4285]

Aslamualikum Sir thanks for these fruitful lectures, but i m facing a problem in using the NCBI blast where in my system only description showed in detail, while all other option showed nothing like graphic and so on. If possible plz reply i m thankful to you sir forever. Regard Mohd Awas (research Scholar) University of Kashmir india

[@Prof.Dr.MuhammadNaveed]

Try again

[@factology2642]

Assalamo alikum sir... how we made algorithm table by blasting....? please help me out

[@nabighafaheem8502]

Assalamualaikum Respected Sir Please guide us about internship program for bs bioinformatics students

[@goodboy-gt3te]

Blast me tools konsy use ho rahy

[@faheemkhan-vj9qg]

good lecture. sir explain ho wo draw phylogenetic tree?

[@Prof.Dr.MuhammadNaveed]

My next lecture 14 is on phylogenetic tree by MEGA

[@uneebashahid5440]

sir I have all the results are almost 100 % , the sequence i used was from GenBank. where I did mistake ? any idea? nucleotide was Saccharomyces cerevicae S288C (PPQ1). And your lecture is very informative,Thanks!!

[@Prof.Dr.MuhammadNaveed]

its impossible have 100% value means you are using same sequence in query

[@uneebashahid5440]

@@Prof.Dr.MuhammadNaveed ok sir thanks!

[@shanzaaslam4826]

Kindly gap penalty bta day r gaps k bary m bta day

[@rizwanhafeez9497]

Sir my question is gpaphics mein similarity kis kay sath show ho rahee hai...Becoz sequence tou aik hi species ka hai yahan pay. Thanks

[@Prof.Dr.MuhammadNaveed]

with all relevant species but different strains

[@sumankarmakar9352]

How to determine that first organism will be selected for further analysis if there were first 5 organism have identity at 100%....and max score,total score all are same....sir please elaborate this....

[@Prof.Dr.MuhammadNaveed]

then means your sequence is copied from these as not possible 5 sequences have 100% similarity

[@hirondevnath2513]

Sir I see a option in BLAST where it shows it can compare two sequences and we get results. From there we can have the variant point but if i want to compare more than 10 sequences to find out the variation point then how it can be done. You know, from blast we can detect the variantion point of two sequences but if i just count and then place it in my paper it’s possible a mistake may happen. As i want to work with 50 sequences, it’s diffcult to run blast everytime and count them. please let me know if any Software or easy solution is available. Take love from Bangladesh. Thanks in advance.

[@Prof.Dr.MuhammadNaveed]

then in simple do run clustalW

[@hirondevnath2513]

@@Prof.Dr.MuhammadNaveed Sir it always shows command timeout.

[@aaaa5nfgfghf]

sir, plz make a video on how use MODELLER software.

[@Prof.Dr.MuhammadNaveed]

noted sure

[@rabiahabib883]

How we trimm sequence

[@namrahqaisar9945]

how to know the difference using two different scoring schemes while using blast

[@Prof.Dr.MuhammadNaveed]

It depend upon %age identity based and which give better result use it

[@muhammadayazfaridshah3694]

sir, kindly ye btain k cancer genes r healthy person k genes kasy download kar skty ham ??? NCBI sy cancer k to mil jaty but healthy person k ni mil rahy mujy.

[@Prof.Dr.MuhammadNaveed]

all from NCBI GenBank just look gene name from literature then search on NCBI

[@muhammadayazfaridshah3694]

@@Prof.Dr.MuhammadNaveed kindly guide me. I have no idea about that. JazakAllah

[@user-lg4yo8me6j]

assalam o alikum- sir mujhy ye samjh nhi aya k" stain novel" hony sy kya murad h?

[@Prof.Dr.MuhammadNaveed]

means every day new strain are discovered in word means new bacteria so NCBI is first method to confirm this novelty on the basis of similarity ok

[@user-lg4yo8me6j]

Ok thank you so much

[@sarimaziz3374]

KINDLY make video on CARD. DATABASE

[@Prof.Dr.MuhammadNaveed]

noted

[@sarimaziz3374]

@@Prof.Dr.MuhammadNaveed sir try to make full sessions on it. I am currently working on it as student but i think the way you explain stuff would be better . Jazakallaha

[@zainabwaheed3606]

I want to ask one question that how we know that the query sequence is novel?

[@Prof.Dr.MuhammadNaveed]

if percentage identity and query coverage are less than 85% then do sequencing again for novelty

[@zainabwaheed3606]

Thank you sir Please guide that how to do sequencing again

[@zainabwaheed3606]

If percentage identity is 84% and Query Cover is 94% then it is novel or not?

[@poonamkumari-nn8yg]

Good evening Sir....I have done sequencing of Mitochondrial COX1 gene of insect specimens. How can I submit these sequences to NCBI.....

[@Prof.Dr.MuhammadNaveed]

watch my lecture on sequence submission on NCBI

[@gopalkalwan7465]

Dear sir i am doing genome wide analysis of gene family and I got 58 hits. What will be the criteria of filteration of these hits for validation purpose or i have to select all

[@Prof.Dr.MuhammadNaveed]

pick hits which have more than 97% query coverage and percentage identity

[@muhammadshahab1421]

Sir can we convert the sequence into 4 Letter idea of protein

[@Prof.Dr.MuhammadNaveed]

no dear

[@gloryparmar1904]

sir, how we can submit SNP to dbSNP or clinvar? can you plz help?

[@Prof.Dr.MuhammadNaveed]

The method is same as you submit your sequence on NCBI GenBank. You need to sign in first and then there is a submission option. You will submit your sequence there and and a specific accession number will be allotted to you.

[@user-bz9xn3rm1f]

Sir , Articles, Research paper Aur publications ma kia frq hy

[@Prof.Dr.MuhammadNaveed]

all same but in publication there are both research and review articles

[@iqralone9785]

sir after getting fasta sequencing of my sample i got 93% query coverage and 97% identity and the species was polaromonas glacialis...is it reliable

[@Prof.Dr.MuhammadNaveed]

yes more than 90% reliable

[@payalprasad5614]

sir what is the the meaning of e value?could you please elaborate

[@Prof.Dr.MuhammadNaveed]

excepted value error mean of query coverage and % identity

[@poonamkumari-nn8yg]

Good evening Sir...I have made mistake in gene bank submission.I have to write in the data isolate instead of voucher specimen.Can I edit my gene bank records ...please help Sir..

[@Prof.Dr.MuhammadNaveed]

yes you can delete and add new detail

[@poonamkumari-nn8yg]

@@Prof.Dr.MuhammadNaveed but...how I can edit...please help...sir

[@nisharaj8967]

Hello sir, Hope you are doing good, I would like to learn Primer design and sequence analysis.

[@Prof.Dr.MuhammadNaveed]

sure and firs watch my lectures on it

[@ashirali195]

sir what is the meaning of rid parameters must be applied when i download the all sequnces from blast?

[@Prof.Dr.MuhammadNaveed]

elaborate your query

[@sanasheikh5033]

How to check bit score ?

[@Prof.Dr.MuhammadNaveed]

The bit score, S', is derived from the raw alignment score, S, taking the statistical properties of the scoring system into account. Because bit scores are normalized with respect to the scoring system, they can be used to compare alignment scores from different searches.

[@hussain223]

Could you speak English, please because I am a foreign researcher

[@Prof.Dr.MuhammadNaveed]

Dear will share in Eglish version after few months as first to complete in native language ok

[@healthlinepakistan3157]

sir how to check SNP rs numbers of stroke.plz guide

[@Prof.Dr.MuhammadNaveed]

look my lecture on mutation identification

[@hamzarehman9913]

Informative lecture. Can I get your contact Dr Sb. Please reply me

[@ambreeniqbal6396]

Sir kindly Chau fasman Py aik lecture plz

[@Prof.Dr.MuhammadNaveed]

sure and noted

[@shabinakhan6930]

Hello sir how to find similarities in genome of two viruses

[@Prof.Dr.MuhammadNaveed]

BY BLAST for sequence similarities

[@oldisgold-nc1zh]

Sir uniquely identified species kese select kare?

[@Prof.Dr.MuhammadNaveed]

use type strain

[@shahnazsalamat1271]

If i can not find any similarity about my protein than what does that mean?

[@Prof.Dr.MuhammadNaveed]

Your sequences are not good or have addition of other sequence or sequencing error

[@shahnazsalamat1271]

@@Prof.Dr.MuhammadNaveed if they have addition of other sequence than what should do ?

[@Prof.Dr.MuhammadNaveed]

Removed it by Bioedit or check via clustalw

[@VivoVivo-ne3qt]

AoA! Sir how to find homozygous and heterozygous through sequence

[@Prof.Dr.MuhammadNaveed]

only possible by phylogentic tree

[@AartiYadav-ns8db]

Sir unknown sequence ko known sequence se kaise match karna h

[@Prof.Dr.MuhammadNaveed]

Blast do this you just need to paste the sequence

[@AartiYadav-ns8db]

@@Prof.Dr.MuhammadNaveed ok

[@nehanadeem8988]

Sir what E value depicts in blast output?

[@muhammadzarghamali4804]

It is significance value similar to those in statistics (P value).

[@iqraafzal4736]

Awsome👍

[@sabgaminggamer2256]

can i do ms computer science after bs in bioinformatics.

[@Prof.Dr.MuhammadNaveed]

Yes dear possible

[@sabgaminggamer2256]

@@Prof.Dr.MuhammadNaveed thanks sirrr.... in which university do you are studing!!

[@Prof.Dr.MuhammadNaveed]

@@sabgaminggamer2256 Teaching in University of Central Punjab, Lahore

[@sabgaminggamer2256]

@@Prof.Dr.MuhammadNaveed great.... well I'm a student of bs in bioinformatics first semester...but i love programming.... like python... artificial intelligence . machine learning... etc.. so... i wanna confirm that from you... ms in computer science...

[@sabgaminggamer2256]

plz make. a tour to our university... which is gcu faisalabad. thanks

[@dr.muhammadimran5501]

Slow down your speed

[@Prof.Dr.MuhammadNaveed]

sure

[@albertescamilla]

I have no idea what you're saying.

[@Prof.Dr.MuhammadNaveed]

very soon you can understand in english version

[@divyanikhode8693]

so fast talking nothing understand

[@Prof.Dr.MuhammadNaveed]

Noted dear and will slow down