GloNeuro

Sequence tools

Tools for the analysis of DNA, RNA and protein sequences. A sequence from NCBI, Ensembl, UniProt or a laboratory’s own sequencing is entered once and can then be analysed with each tool.

Sequence input

The input is one sequence, or several in FASTA format, each beginning with a line that starts with “>”. Spaces, line breaks and position numbers are removed, and the type of sequence (DNA, RNA or protein) is detected automatically.

All calculations run in the browser. Sequences are not sent to GloNeuro or to any third party and are not saved on a server or in browser storage; the text is held only while the page is open.

The example sequences are random and synthetic; they are not real genes or proteins.

The tools on this page need JavaScript, which appears to be switched off in this browser.

DNA and RNA

Tools for nucleotide sequences: strands and transcription, composition, translation and reading frames, CpG islands, restriction enzymes, primers and PCR, and motifs.

The sequence of the opposite strand, read 5′ to 3′, with IUPAC codes handled.

DNA to RNA and RNA back to complementary DNA, from either strand.

Base counts, GC content and skew, and a sliding-window plot of GC content.

Molar mass of single- or double-stranded, linear or circular nucleic acid, and picomoles per microgram.

All six frames, with the standard or the vertebrate mitochondrial genetic code.

Possible protein-coding stretches on both strands, drawn on a map.

How often each of the 64 codons is used in a coding sequence.

CpG depletion and a scan for CpG islands by two published sets of criteria.

Sites of 36 common enzymes, cut positions, fragment sizes and a virtual gel.

Tm by three methods, including nearest-neighbour thermodynamics, and simple checks for problems.

Where two primers bind a template, and the size and sequence of the products.

Every match to a short pattern on both strands, with IUPAC codes allowed.

Proteins

Tools for amino-acid sequences: composition, molecular weight, extinction coefficient and isoelectric point, hydropathy and possible membrane-spanning segments, protease digests and peptide masses, PROSITE motifs, a disorder indicator, amino-acid substitutions and back-translation.

Amino-acid composition, average and monoisotopic mass, absorbance at 280 nm, isoelectric point and charge with a choice of pKa values, GRAVY, instability and aliphatic indices.

Kyte–Doolittle hydropathy in a sliding window, with the stretches that may span a membrane marked: a simple prediction.

Trypsin, Lys-C, chymotrypsin, Asp-N, Glu-C and cyanogen bromide, with missed cleavages and monoisotopic peptide masses for mass spectrometry.

User-defined PROSITE-syntax patterns and classic examples such as the N-glycosylation sequon, with the number of matches expected by chance.

Whether the composition resembles that of natively unfolded proteins, for the whole protein and in a sliding window, with links to real disorder and secondary-structure predictors.

The change in mass, pI, charge and hydropathy, the BLOSUM62 score and the classes of the two residues.

Every DNA sequence that could encode the protein, written with IUPAC codes, in an inclusive or an exact form.

Alignment and comparison

Aligning two or more sequences and measuring how similar they are.

Two DNA or protein sequences aligned end to end with affine gap penalties, BLOSUM or PAM250 scoring, identity, similarity and a dot plot.

The best-matching region shared by two sequences, such as a common domain, with its score and a dot plot.

A small set of sequences aligned along a guide tree, coloured by residue, with conservation, consensus, FASTA and CLUSTAL export.

The percentage of identical positions for every pair of sequences, as a shaded table.

Evolution and phylogeny

Relationships between sequences from different genes or species.

p-distance, Jukes–Cantor, Kimura 2-parameter, Poisson and Kimura protein distances for every pair, with CSV and PHYLIP export.

A distance tree drawn as a phylogram or cladogram, rooted at the midpoint or on an outgroup, with bootstrap percentages and Newick export.

dN, dS and their ratio for two aligned coding sequences, by the Nei–Gojobori method, with every differing codon listed.

Mutations and variants

Making a change to a coding sequence and describing the differences between two sequences, with the predicted effect on the protein.

The codon and amino acid before and after, and whether the change is synonymous, missense, nonsense, a frameshift or start-loss.

Every difference from a reference, in simplified HGVS-style c. notation, with its predicted effect on the protein.

Where to find sequences

Public databases hold the sequences of genes, transcripts and proteins from thousands of species, free to use. Each can give a sequence in FASTA format, a plain-text format in which a line beginning with “>” names the sequence and the following lines hold the sequence itself. A downloaded FASTA file can be pasted or opened with Open a file.

NCBI Nucleotide

The US National Center for Biotechnology Information collects nucleotide sequences submitted by laboratories worldwide (shared daily with the European and Japanese archives, ENA and DDBJ). Search by gene name and organism, or by accession number. On a record, choose FASTA at the top to see the sequence in FASTA format and copy it, or use Send to → File → FASTA to download it. To take part of a record, such as the coding sequence, follow the CDS link in the list of features.

NCBI Gene

A summary page for each gene, with its location, its transcripts and its proteins. The section on NCBI reference sequences lists the curated transcripts (accessions beginning NM_) and proteins (NP_). Open one to reach its Nucleotide or Protein record, then choose FASTA.

RefSeq

NCBI’s Reference Sequence collection gives one curated, non-redundant sequence for each molecule: NC_ for chromosomes, NM_ for mRNAs, NR_ for non-coding RNAs and NP_ for proteins; XM_ and XP_ are computer-predicted models. An NM_ or NP_ record is the preferred reference for describing a variant.

Ensembl

A genome browser maintained by EMBL-EBI. Search for a gene, choose a transcript, then open Exons, cDNA or Protein under Sequence and use Download sequence for FASTA. Some downloads mark repeats or flanking sequence in lower-case letters; the tools convert all letters to capitals.

UniProt

The Universal Protein Resource. Reviewed entries (Swiss-Prot) are curated by experts; unreviewed entries (TrEMBL) are annotated automatically. On an entry, the Sequence section offers the sequence to copy, and Download gives it in FASTA format. Isoforms have their own sequences.

RCSB Protein Data Bank

Three-dimensional structures of proteins and nucleic acids solved by X-ray crystallography, cryo-electron microscopy or NMR. On a structure page, Download Files → FASTA Sequence gives the sequences of the chains. These are the molecules that were studied, which may be fragments, carry tags or contain deliberate mutations.

AlphaFold Protein Structure Database

Structures predicted by AlphaFold (Jumper et al., 2021) for most proteins in UniProt, searchable by UniProt accession, with a confidence score for every residue (Varadi et al., 2022). The sequence of each prediction is that of its UniProt entry, so the FASTA sequence is best taken from UniProt.

What needs a database or a server

The tools hold no database, so every result is calculated from the sequences entered. Some questions can be answered only by comparing a sequence with large collections of data or by running large models; public services for these purposes are listed below.

Searching databases for similar sequences

Finding the known genes or proteins that resemble a sequence requires a search of a whole database, with BLAST (Altschul et al., 1990) at NCBI BLAST or the sequence search services of EMBL-EBI.

Gene Ontology and enrichment analysis

Testing which functions or pathways are over-represented in a list of genes requires the annotations of every gene, which the Gene Ontology resource (Ashburner et al., 2000) provides.

Predicting a protein’s structure

Structure prediction requires large trained models and databases of related sequences. Existing predictions are available in the AlphaFold Protein Structure Database and solved structures in the Protein Data Bank.

Checking that primers are specific

Whether primers bind anywhere else in a genome can be checked only against the genome itself, with NCBI Primer-BLAST.

Interpreting a variant

Whether a variant has been seen before, how common it is and whether it is linked to disease are recorded in ClinVar, dbSNP and gnomAD. The full rules for describing variants are at hgvs-nomenclature.org.

References

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The calculations were checked against Biopython (Cock et al., 2009) on random sequences. The genetic codes are NCBI’s translation tables 1 and 2. Results are intended for teaching and planning; important results should be confirmed with a second tool.

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