Overview. Sixpack is described succinctly on its website:
Sixpack reads a DNA sequence and outputs the three forward and (optionally) three reverse translations in a visual manner.
In each reading frame, sixpack will also return a list of proteins that can be translated, which is done by enumerating open reading frames (ORFs). For virus discovery, sixpack can then be used to enumerate the proteins encoded by a viral genomic sequence.
Tutorial objective. We will use sixpack to conduct a simple sanity check, namely, to recover the palmprint in a viral genomic sequence.
The EMBOSS sixpack webtool can be accessed through the EMBL-EBI website.
We note that other APIs are available (see the sixpack documentation for further details).
The main input box of sixpack STEP 1 requires a DNA or RNA sequence,
given in any one of the supported formats, which include FASTA, GCG, EMBL, and GenBank.
Our DNA sequence is an assembled contig from the SRA run SRR17250291 that was detected to contain an RdRp palmprint.
The detected palmprint sequence (with A, B, C motifs annotated) is:
LEIDISKYDKSQGALVLEFEIKMMRLFGVDEELCFYWRKAHECTTLWDRLNGVKCKVLYQRKSGDASTYLGNTLFLMAVICCLFDLSRVTMGLFSGDDSLI
|----A-----| |------B-----| |---C--|
For this tutorial, we will provide the DNA sequence in a FASTA file, uploaded at tools/img/sixpack/contig.fasta :
$ head tools/img/sixpack/contig.fasta
>SRR17250291_NODE_133
CACAGGACAGAACAACACAAATTTGGCAGGAATCACATGATAACGGTTCACGCTCTTTAG
TTAGAGTTTCACCTTTTCATTGTCTCTTTTGGATTCTGTCTGTTTGGTGCTTAGGCCCTG
GCGGACATTTCTTGAGAATTTCTGTTTTCTTTGACCTTTAACGGTGCGTTTAGCTCGTTT
TTGGTCTCTGGATTTGGATTATTCTTTGGATTTTTCTTTAAGAGATTTCATTTTCCTTTT
TCAATTTTTCATTTTCAAATTTCTAATTTAAATTTTCATTTTCATTGAAACATTGTTTTG
GATTTTCTTTCATAGAACCGTTATCATGTGATTCCTGCCAAATTTGTGTTGTTCTGTCCT
GTGCCGGGTGTTCTTTTGAACATTAGGTGCAATATTTACCTTTTGTGTTCTGTGTTCATA
TTTACTTTATTTTGTATATATTTATATTTTCTTTTGTATATATATATTTTGTATTATTAT
TTTGTGCAATATTTTGTGTTTTTCTGGTTCCTAATATCACCATGTCTGTGTCTCGCTTGCTo use sixpack, in the STEP 1 box, either (1) copy and paste the contents of contig.fasta into the prompt
or (2) upload the file itself. Next, hit the Submit button in the STEP 3 box to launch the tool.
The STEP 2 box contains some parameters that can be toggled. We use the default settings, but
we document them for completeness below:
- Codon table: the genetic code to use for translation (see the NCBI documentation)
- First ORF: whether input sequence prefixes should be considered as ORFs, disregarding the minimum ORF size
- Last ORF: whether input sequence suffixes should be considered as ORFs, disregarding the minimum ORF size or if they end in a stop codon
- Reverse: whether to return translations for the reverse-compliment of the input sequence
- ORF min. size: the minimum protein size for returned ORFs
Further details are given in the sixpack documentation.
The webtool will redirect you to a new page with a couple of tabs near the top.
We are primarily interested in the Result Summary (which you should be on to begin)
and Tool Output tabs, and describe them in the following. The results
can be downloaded by clicking the Download ... button underneath the tabs.
This page begins by visualizing the input sequence and (optionally) its reverse compliment
and the codons obtained by translating the sequences in various reading frames. The following
are the first few lines of the page (line numbers LX added for reference):
H R T E Q H K F G R N H M I T V H A L * F1 (L1)
T G Q N N T N L A G I T * * R F T L F S F2 (L2)
Q D R T T Q I W Q E S H D N G S R S L V F3 (L3)
1 CACAGGACAGAACAACACAAATTTGGCAGGAATCACATGATAACGGTTCACGCTCTTTAG 60 (L4)
----:----|----:----|----:----|----:----|----:----|----:----| (L5)
1 GTGTCCTGTCTTGTTGTGTTTAAACCGTCCTTAGTGTACTATTGCCAAGTGCGAGAAATC 60 (L6)
X L V S C C L N P L F * M I V T * A R * F6 (L7)
X C S L V V C I Q C S D C S L P E R E K F5 (L8)
V P C F L V F K A P I V H Y R N V S K L F4 (L9)
The sequence is shown in L4 and its reverse compliment in L6. The flanking numbers 1 and 60
indicate that we are looking at the 1-st to 60-th (inclusive) bps of the input sequence. Each row
above and below gives the amino acids (AAs) obtained by translating the input sequence in a specific reading frame,
labelled FX for "frame X".
F1-3 correspond to the forward sequence while F4-6 correspond to the reverse-compliment.
Each AA is aligned with the first nucleotide of the codon from which it was translated. For example, the
first AA H of F1 was produced from the CAC codon, and the fourth AA N of L2 was
produced from AAC. We clarify this with bars:
H R T E Q H K F G R N H M I T V H A L * F1 (L1)
|T G Q N N T N L A G I T * * R F T L F S F2 (L1)
| | (L3 omitted)
1 CACAGGACAGAACAACACAAATTTGGCAGGAATCACATGATAACGGTTCACGCTCTTTAG 60 (L4)
Note that a * denotes a stop codon. For F4-6, care must be taken since these
are translations of the sequence's reverse compliment. Hence, L6-9 should be read from
right to left for all intents and purposes. For example, the S in the following is produced by the
codon TCC, not CCT or TGT:
1 GTGTCCTGTCTTGTTGTGTTTAAACCGTCCTTAGTGTACTATTGCCAAGTGCGAGAAATC 60 (L6)
| (L7 omitted)
X C S L V V C I Q C S D C S L P E R E K F5 (L8)
Finally, at the very end of the page, there will also be some summary statistics that count the number of ORFs in each reading frame:
Minimum size of ORFs : 1
Total ORFs in frame 1 : 139
Total ORFs in frame 2 : 408
Total ORFs in frame 3 : 20
Total ORFs in frame 4 : 217
Total ORFs in frame 5 : 144
Total ORFs in frame 6 : 122
Total ORFs : 1050
This page contains a FASTA file enumerating the proteins translated from all possible ORFs in all
of the three or six reading frames in the Result Summary. Indeed, we find that our palmprint sequence
is contained in an entry (although split across two lines):
>SRR17250291_NODE_133_3_ORF6 Translation of SRR17250291_NODE_133 in frame 3, ORF 6, threshold 1, 3009aa
VQYLPFVFCVHIYFILYIFIFSFVYIYFVLLFCAIFCVFLVPNITMSVSRLRSGFCYVKW
...
SLDKLEIDISKYDKSQGALVLEFEIKMMRLFGVDEELCFYWRKAHECTTLWDRLNGVKCK <-
VLYQRKSGDASTYLGNTLFLMAVICCLFDLSRVTMGLFSGDDSLIVGEGINHDRNQECAL <-
LFNLESKFFRSYKYMYFCSKFLIPYAGGFKFIPDPVKLITKMGRHDLVNFDHVEEYRVSM
CDLTKDFSNGIINEILSEAINERYLCYGNHSMVFSCIHNLTKNSKLFRELYYTNEGDVLC
MDVSRPSLD
The entry title aptly indicates the reading frame (frame 3), ORF number (6), and AA length of the
translated sequence (3009aa).
That's it, we have used EMBOSS sixpack to recover the palmprint in our viral contig! In summary, sixpack translates a DNA sequence across multiple reading frames, and outputs the results as a human-interpretable text page and a FASTA file of translated ORFs. These can be used to better understand what proteins may be encoded in a viral genomic sequence.