Egon A. Ozer, MD PhD ([email protected])
Ramon Lorenzo Redondo, PhD ([email protected])
Activate the conda environment:
conda activate annotation
WARNING: If you are using a Mac, the Conda version of prokka is too buggy for us to use. Instead, use the following commands to install prokka:
If you have HomeBrew installed:
brew install brewsci/bio/prokka
sudo cpan install Bio::SearchIO::hmmerIf you don't have HomeBrew:
sudo cpan Time::Piece XML::Simple Digest::MD5 Bio::Perl Bio::SearchIO::hmmer
git clone https://github.com/tseemann/prokka.git $HOME/prokka
$HOME/prokka/bin/prokka --setupdbGenome annotation is the process of determining the key features of the genome. This usually incldes identifying gene sequences, but can also include other features such as RNA encoding regions, signal peptides or other important features. The process of annotation is a bit different whether you are working with eukaryotes, prokaryotes, or viruses, but there are a number of programs and applications available to help you with this.
One of the best annotation applications for bacteria and archaea is the NCBI Prokaryotic Genome Annotation Pipeline (PGAP. Automatic PGAP annotation can be requested when depositing genome sequences in NCBI and is ultimately their preferred method of annotation. NCBI has made a version of PGAP available for running on demand on your local computer (https://github.com/ncbi/pgap), but it is a bit complex to install and maintain.
If you want quick and useful annotations of bacterial, archeal, or viral genomes you've sequenced and assembled, prokka is a pretty good choice.
Commands
prokka \
--outdir GAS_annotation \
--prefix GAS_example \
--genus Streptococcus \
--species pyogenes \
--proteins reference/GAS_NGAS638.gbk \
--compliant \
--cpus 0 \
GAS_assembly/contigs.fastaSettings
| Setting | Descripton |
|---|---|
--outdir |
Name of the directory the output files will go into. You'll get an error if this directory already exists |
--prefix |
Name that all of the output files will begin with |
--genus |
Genus ID of the organism |
--species |
Species ID of the organism |
--proteins |
Annotated reference sequence(s) for prokka to use to identify similar proteins in the assembled sequence. Not required, but if you have a closely related reference, this will result in more useful annotations of your assembly. |
--compliant |
Does some extra filtering to make the annotation "complaint" with NCBI requirements including removing contigs shorter than 200 bp |
--cpus |
Number of compute cores to use. Setting to '0' allows it to detect how many cores your computer has available and use them all |
See https://github.com/tseemann/prokka more detail on available settings.
Outputs
Adapted from https://github.com/tseemann/prokka. Follow the link to see the full list of output files.
| Extension | Description |
|---|---|
| .fna | Nucleotide FASTA file of the input contig sequences. |
| .faa | Protein FASTA file of the translated CDS sequences. |
| .ffn | Nucleotide FASTA file of all the prediction transcripts (CDS, rRNA, tRNA, tmRNA, misc_RNA) |
| .gbk | This is a standard Genbank file. If the input to prokka was a multi-FASTA, then this will be a multi-Genbank, with one record for each sequence. |
BLAST (Basic Local Alignment Search Tool) is one of the most important and most widely used bioinformatic tools. The original publication by Altschul et al "Basic local alignment search tool" has been cited almost 68,000 times since its publication in 1990. As a tool for searching nucleotide or protein sequences, BLAST is uncontested.
The web version of the BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) has many advantages including a diversity of databases for searching, graphical output, and links to more detailed information on hits, but can suffer from long wait times and limits searchable sequence lengths. Often having the option to perform some BLAST searches on your own computer can be quick and efficient.
Let's say we're interested in whether our assembled genome sequence contains the fibronectin-binding surface protein FbaA, a protein associated with some strains of Streptococcus pyogenes causing skin infections. We can use nucleotide BLAST (blastn) to search our assembly for the nucleotide sequence of the fbaA gene or we can use protein BLAST (blastp) to search the protein sequences from our prokka annotation. BLAST programs have a choice of outputs that can make our searches more efficient, depending on what we're looking for.
Commands
A. Search the genome assembly for the fbaA gene sequence. This will generate the default pairwise output format.
blastn \
-query reference/fbaA.fasta \
-subject GAS_assembly/contigs.fasta B. Search the prokka annotation for the FbaA protein sequence. Here we'll generate a tabular output format instead.
blastp \
-query reference/FbaA.faa \
-subject GAS_annotation/GAS_example.faa \
-outfmt 6Settings
| Setting | Descripton |
|---|---|
-query |
Query sequence in fasta format. This is the sequence you are searching FOR. |
-subject |
Subject sequence in fasta format. This is the sequence you are searching IN. |
-outfmt |
Output format. The default is '0', i.e. pairwise. '6' produces standard tabular output. |
For more information on settings and how to further customize output formats, run the blast program with the -help setting, i.e.
blastn -helpOutputs
By default the output of the blast commands will be printed to the screen. If you want to output the results into a file instead, either use the -out setting to give an output file or just direct the output to a file using >.
A. Output of blastn nucleotide search with pairwise formatting:
BLASTN 2.12.0+
Reference: Zheng Zhang, Scott Schwartz, Lukas Wagner, and Webb
Miller (2000), "A greedy algorithm for aligning DNA sequences", J
Comput Biol 2000; 7(1-2):203-14.
Database: User specified sequence set (Input: GAS/contigs.fasta).
32 sequences; 1,802,596 total letters
Query= AB040536.1:142-1209 Streptococcus pyogenes fbaA gene for
fibronectin-binding protein, complete cds
Length=1068
Score E
Sequences producing significant alignments: (Bits) Value
NODE_4_length_123086_cov_28.457640 1973 0.0
> NODE_4_length_123086_cov_28.457640
Length=123086
Score = 1973 bits (1068), Expect = 0.0
Identities = 1068/1068 (100%), Gaps = 0/1068 (0%)
Strand=Plus/Plus
Query 1 ATGCGTAGAGCAGAAAATAACAAACACAGCCGCTATTCCATTCGCAAACTGAGCGTTGGG 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 121737 ATGCGTAGAGCAGAAAATAACAAACACAGCCGCTATTCCATTCGCAAACTGAGCGTTGGG 121796
Query 61 GTAACGAGTATAGCAATTGCGAGTCTCTTTTTAGGAAAGGTTGCCTATGCCGTAGATGGC 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 121797 GTAACGAGTATAGCAATTGCGAGTCTCTTTTTAGGAAAGGTTGCCTATGCCGTAGATGGC 121856
Query 121 ATCCCTCCAATCTCTCTTACTCAAAAGACTACAGCCACTACATCAGAAAATTGGCATCAT 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 121857 ATCCCTCCAATCTCTCTTACTCAAAAGACTACAGCCACTACATCAGAAAATTGGCATCAT 121916
Query 181 ATTGATAAGGATGGCCTTATTCCTTTAGGTATAAGCTTAGAAGCTGCCAAAGAGGAATTT 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 121917 ATTGATAAGGATGGCCTTATTCCTTTAGGTATAAGCTTAGAAGCTGCCAAAGAGGAATTT 121976
Query 241 AAAAAAGAAGTAGAAGAATCACGTTTATCTGAAGCACaaaaagaaacgtataaacaaaaa 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 121977 AAAAAAGAAGTAGAAGAATCACGTTTATCTGAAGCACAAAAAGAAACGTATAAACAAAAA 122036
Query 301 attaaaaCTGCACCAGACAAAGATAAGCTATTATTCACGTATCATAGTGAGTATATGACA 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 122037 ATTAAAACTGCACCAGACAAAGATAAGCTATTATTCACGTATCATAGTGAGTATATGACA 122096
Query 361 GCCGTTAAGGATCTTCCAGCGTCTACTGAGTCTACTACTCAGCCAGTTGAGGCACCCGTG 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 122097 GCCGTTAAGGATCTTCCAGCGTCTACTGAGTCTACTACTCAGCCAGTTGAGGCACCCGTG 122156
Query 421 CAGGAGACACAGGCATCAGCTTCAGATTCGATGGTGACAGGTGATTCAACATCAGTTACG 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 122157 CAGGAGACACAGGCATCAGCTTCAGATTCGATGGTGACAGGTGATTCAACATCAGTTACG 122216
Query 481 ACTGATTCTCCTGAGGAAACCCCATCTTCGGAAAGTCCAGTGGCCCCAGCTTTATCTGAG 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 122217 ACTGATTCTCCTGAGGAAACCCCATCTTCGGAAAGTCCAGTGGCCCCAGCTTTATCTGAG 122276
Query 541 GCTCCAGCTCAACCAGCTGAGAGTGAGGAACCTTCAGTAGCAGCATCTTCTGAGGAAACC 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 122277 GCTCCAGCTCAACCAGCTGAGAGTGAGGAACCTTCAGTAGCAGCATCTTCTGAGGAAACC 122336
Query 601 CCATCTCCATCAACTCCAGCAGCCCCATCAACTCCAGCGGCTCCAGAAACTCCTGAAGAA 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 122337 CCATCTCCATCAACTCCAGCAGCCCCATCAACTCCAGCGGCTCCAGAAACTCCTGAAGAA 122396
Query 661 CCAGCAGCTCCATCTCAACCAGCTGAGAGTGAAGAATCTTCAGTAGCAGCTACGACAAGC 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 122397 CCAGCAGCTCCATCTCAACCAGCTGAGAGTGAAGAATCTTCAGTAGCAGCTACGACAAGC 122456
Query 721 CCGTCTCCATCAACTCCAGCTGAATCAGAGACTCAGACGCCACCAGCTGTTACTAAAGAC 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 122457 CCGTCTCCATCAACTCCAGCTGAATCAGAGACTCAGACGCCACCAGCTGTTACTAAAGAC 122516
Query 781 TCTGATAAGCCATCTTCAGCAGCTGAAAAACCAGCAGCCTCTTCACTTGTTTCAGAACAA 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 122517 TCTGATAAGCCATCTTCAGCAGCTGAAAAACCAGCAGCCTCTTCACTTGTTTCAGAACAA 122576
Query 841 ACCGTTCAACAACCAACTTCAAAGAGATCTTCTGATAAAAAAGAAGAGCAAGAACAGTCT 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 122577 ACCGTTCAACAACCAACTTCAAAGAGATCTTCTGATAAAAAAGAAGAGCAAGAACAGTCT 122636
Query 901 TACTCTCCAAATCGCTCATTGTCAAGACAGGTTAGGGCCCATGAGTCAGGTAAGTACTTG 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 122637 TACTCTCCAAATCGCTCATTGTCAAGACAGGTTAGGGCCCATGAGTCAGGTAAGTACTTG 122696
Query 961 CCTTCAACAGGTGAAAAAGCACAGCCACTCTTTATAGCTACTATGACTTTGATGTCTCTA 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 122697 CCTTCAACAGGTGAAAAAGCACAGCCACTCTTTATAGCTACTATGACTTTGATGTCTCTA 122756
Query 1021 TTTGGCAGTCTTTTAGTCACaaaacgccaaaaagaaactaaaaaaTAG 1068
||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 122757 TTTGGCAGTCTTTTAGTCACAAAACGCCAAAAAGAAACTAAAAAATAG 122804
Lambda K H
1.33 0.621 1.12
Gapped
Lambda K H
1.28 0.460 0.850
Effective search space used: 1884779032
Database: User specified sequence set (Input: GAS/contigs.fasta).
Posted date: Unknown
Number of letters in database: 1,802,596
Number of sequences in database: 32
Matrix: blastn matrix 1 -2
Gap Penalties: Existence: 0, Extension: 2.5
B. Tabluar ('6') output of blastp search:
BAB62098.1 OIOADCLO_01010 100.000 355 0 0 1 355 1 355 0.0 694
BAB62098.1 OIOADCLO_01006 30.357 112 65 4 4 102 2 113 1.93e-04 38.5
BAB62098.1 OIOADCLO_01006 45.000 40 21 1 315 353 445 484 0.003 35.0
BAB62098.1 OIOADCLO_00241 26.174 149 67 5 203 350 184 290 0.003 34.3
BAB62098.1 OIOADCLO_00960 25.974 77 36 2 59 124 350 416 0.051 31.2
BAB62098.1 OIOADCLO_01690 40.000 35 20 1 320 353 183 217 0.084 29.6
BAB62098.1 OIOADCLO_00464 45.833 24 13 0 8 31 3 26 0.40 28.1
BAB62098.1 OIOADCLO_00464 44.737 38 17 2 318 354 1612 1646 4.2 25.0
BAB62098.1 OIOADCLO_01255 47.059 34 17 1 318 350 312 345 0.67 27.3
BAB62098.1 OIOADCLO_01064 42.308 26 15 0 247 272 7 32 1.7 25.4
BAB62098.1 OIOADCLO_00866 25.166 151 89 5 79 229 213 339 2.7 25.4
BAB62098.1 OIOADCLO_01637 33.333 30 20 0 2 31 155 184 4.6 24.6
Tabular standard output format:
- Query ID
- Subject ID
- Percent identity
- Alignment length
- Number of mismatches
- Number of gaps opened
- Start of the alignment on the query sequence
- End of the alignment on the query sequence
- Start of the alignment on subject sequence
- End of the alignment on the subject sequence (if subject start coordinate is greater than subject end coordinate, this indicates the query is aligned on the minus strand of the subject sequence.
- Expect value (E-value)
- Bit score
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