It's a simple question but I've come across many people who have this question, is the reference genome Positive of Negative strand? Indeed, I've had heated arguments over the same issue.

So here's to putting all those questions to rest.

Is the reference genome a positive or a negative strand? And why so.

  • $\begingroup$ Koustav. Thanks for your effort; it is quite commendable. IMO Q/A such as this which are intended to serve as a reference can be made a community wiki. It makes posts easily editable without much rep requirement; flipside is that you don't get reputation points. It is just a suggestion and it is up to you whether you want to do that or not. $\endgroup$ – WYSIWYG Aug 3 '15 at 17:56
  • $\begingroup$ Done! I didn't notice that option before! $\endgroup$ – FoldedChromatin Aug 3 '15 at 19:28
  • $\begingroup$ @WYSIWYG — why is this question a community wiki? It is imprecise and based on a false assumption, as my answer makes clear. $\endgroup$ – David Sep 10 '18 at 20:27
  • $\begingroup$ @David The post sounded like a popular question for which an authoritative answer can be provided and therefore I proposed CW. BTW, the assumption is not entirely incorrect. $\endgroup$ – WYSIWYG Sep 13 '18 at 8:19

First of all, the reference genome strand specificity is referred to as sense (positive strand) or antisense (negative strand). Now let's consider to sequencing data or FASTQ files. When we align reads, a resulting SAM or BAM file has a column specifying strand information, we usually see a + or - strand.

For more background on the strand names here's a somewhat incorrect note from the wiki article under sense (molecular biology)

The strand names actually depend on which direction you are writing the sequence that contains the information for proteins (the "sense" information), not on which strand is on the top or bottom (that is arbitrary). The only real biological information that is important for labeling strands is the location of the 5' phosphate group and the 3' hydroxyl group because these ends determine the direction of transcription and translation. A sequence 5' CGCTAT 3' is equivalent to a sequence written 3' TATCGC 5' as long as the 5' and 3' ends are noted. If the ends are not labeled, convention is to assume that the sequence is written in the 5' to 3' direction. Watson strand refers to 5' to 3' top strand (5' → 3'), whereas Crick strand refers to 5' to 3' bottom strand (3' ← 5').[4] Both Watson and Crick strands can be either sense or antisense strands depending on the gene whose sequences are displayed in the genome sequence database. For example, YEL021W, an alias of URA3 gene used in NCBI database, defines that this gene is located on the 21st open reading frame (ORF) from the centromere of the left arm (L) of Yeast (Y) chromosome number V (E), and that the expression coding strand is Watson strand (W). YKL074C defines the 74th ORF to the left of the centromere of chromosome XI and denotes coding strand from the Crick strand (C). Another confusing term referring to "Plus" and "Minus" strand is also widely used. Whatever the strand is a sense (positive) or antisense (negative), the default query sequence in NCBI BLAST alignment is "Plus" strand.

Although it is correct when it says

  1. Watson = Sense = Plus Strands
  2. Crick = Antisense = Negative Strands

A satisfactory answer/convention is provided/suggested in this PMC article covering the terminology of the Watson and Crick strands

Below are some interesting excerpts from the publication:

The earliest reference that we could find to the "Watson strand" and the "Crick strand" is somewhat tongue-in-cheek and comes from a pair of papers in 1967 by Wacław Szybalski and colleagues. They bound the two DNA strands of phage λ to the synthetic polynucleotide, poly(IG), which has an affinity to cytosine-rich regions. They then separated the two strands by density, which turned out to be determined by the amount of bound poly(IG). In a cesium-chloride density gradient, the strand with more bound poly(IG) was denser and heavier than its complement. Because the "dense" strand was cytosine-rich, Szybalski and colleagues called it the "C strand." Logically, thus, the complementary strand, which was guanine rich, should have been the "G strand." Instead, it was christened the "W strand."

So the authors note that the Strands started on their terminology journey with their roles reversed with the Crick strand specifying the heavy strand (with IG) and the Watson strand specifying the lighter one.

They also note that the present day terminology of the WC model is non-arbitratrily based on the current horizontal drawing convention where one strand is placed on top and the other at the bottom.

They also suggested a typical convention which is followed today. If it was due to their suggestion is a different matter altogether, but beyond the scope of this question.

Given the amount of effort already spent on standardizing such databases, and their influence on other disciplines, we feel that the genomic definition of Watson and Crick strands has the most mass behind it. Specifically, we find that the unambiguous usage of the Saccharomyces Genome Database to be the most useful. Under the first part of our proposal, the centromere is a reference point that divides a chromosome into two arms of unequal length. The chromosome is oriented so that shorter arm is on the left and the longer arm on the right. Furthermore, the top strand has its 5'-end at the left (short-arm) telomere and its 3'-end at the right (long-arm) telomere. This strand is the Watson strand. Similarly, the bottom strand has its 5'-end at the right telomere and its 3' at the left telomere and is the Crick strand. We further propose that "top", "forward", and "plus" be used as synonyms for the Watson strand and "bottom", "reverse", and "minus" for the Crick strand.

and later on...

If it is ultimately impossible to distinguish Watson and Crick strands using biological properties, then we propose that Watson should refer to the stand arbitrarily used as a reference in a database (i.e. the "plus" stand) and the Crick strand should refer to its complement

But let's come back to sequencing data or FASTQ files. When we align reads, a resulting SAM or BAM file has a column specifying strand information, we usually see a + or - strand.

Which suggests that the product originated from either the Watson (positive) or the Crick (negative) strands. For example, a read is actually the reverse complement of the product, but since you do a PCR step during you library preparation, that particular bit of information is lost and therefore the downstream analysis protocols tend to consider the entire loci any single product aligns to.

The difference between strand specific sequencing and non-strand specific sequencing are covered here.

  • 3
    $\begingroup$ The present state of this answer seems to confound terminology appropriate for gene-relative purposes with that appropriate for chromosome-relative purposes. Gene-relative uses Sense/Antisense (or Coding/Template), while chromosome-relative uses Forward/Reverse (or Plus/Minus). A Forward strand will have both Sense and Antisense sequences on it. See Bio_X2Y's answer here for an accurate and concise description. $\endgroup$ – mgkrebbs Sep 27 '16 at 23:44
  • $\begingroup$ I'm sorry I'd go further than @mgkrebbs and say both question and this answer are nonsense. I am not familar with Community wikis, but this should definitely not be some standard reference. I have written an answer explaining why. $\endgroup$ – David Sep 10 '18 at 20:25

There is no such thing as a positive or negative strand for a genome (reference or otherwise), for the simple reason that the genomes of almost all organisms contain genes in both orientations, and hence each strand contains genes the sequence of which are in the sense and the anti-sense direction with respect to the mRNA.

The exception is single-stranded RNA viruses, where a single strand may act as the mRNA. This is where the ‘+’ and ‘–’ nomenclature is mainly used — to distinguish which strand is used in the virus genome.

I have previously addressed this topic in my answers to related questions about sequence direction in databases and reading frames.

Postscript: where do reference genomes begin and on which strand?

To find the answer to this question you should look at the documentation in the database for the particular genome of interest. However, as far as I am aware, the following is generally true. (Others may be able to improve on this.)

  1. In bacterial and plasmid genomes the sequence usually starts from the (single) origin of replication, proceeding in the direction in which replication occurs, written to the right of the origin. The strand represented in the database is the one starting with a 5′ end on the left at this origin.
  2. In eukaryotic genomes that have been well studied there is a recognizable cytochemical assymetry of the chromosomes (in terms of banding of heterochromatin and, in some cases, ‘arms’) and there is a convention to designate one end ‘left’ and one ‘right’. In the reference genomes the sequence starts from the conventially viewed left of the chromosome, presenting the DNA sequence of the strand that has the 5′ end.
  3. In single-stranded RNA viruses — where the ‘+’ and ‘–’ nomenclature is used — the sequence is of the ‘+’, starting at the 5′ end.
  • $\begingroup$ There is however a natural direction depending on the direction of replication. $\endgroup$ – Jack Aidley Sep 10 '18 at 13:21
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    $\begingroup$ @JackAidley — Direction of replication? This may hold for bacteria, but not for eukaryotes. But, although it wasn't strictly the question and the poster is long gone I'veadded something about where the "reference genomes" start from. $\endgroup$ – David Sep 10 '18 at 20:19
  • $\begingroup$ The + and - is a bioinformatics classification. The reference sequence is by default the + and all the genes in the opposite orientation are annotated as -. So while what you say is correct there is a standard reference sequence and it is the + strand for all analysis. $\endgroup$ – WYSIWYG Sep 13 '18 at 8:22
  • $\begingroup$ @WYSIWYG — I see your point that + and – are used in the data files but the poster's question "Is the reference genome a positive?" clearly indicates that this is not what he meant, otherwise the answer is the trivial '+'. And the terms + strand and – strand were used in molecular virology before nucleic acid sequencing was invented, so although I have written programs to read GenBank files and am familiar with this designation, I never heard them refered to as + strand. The other answer also assumes that the poster is talking about "sense" strands, although he goes off on a tangent rather. $\endgroup$ – David Sep 13 '18 at 15:52

The + and - is a bioinformatics classification. The reference sequence is by default the + and all the genes in the opposite orientation are annotated as -.

For linear eykaryotic chromosomes, the reference genome sequence is in the orientation of the chromosome (based on older cytogenetic assignment; usually the short arm is 5').

For prokaryotes, I guess the origin of replication is the beginning of the reference genome (Eisen et al, 2000).

  • $\begingroup$ I would replace "bioinformatics classification" by "data file designation". GenBank files and the like are human-readable, so they have strictly nothing to do with informatics, and I don't think there is any classification involved. $\endgroup$ – David Sep 13 '18 at 15:55

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