# Chromosome identification methods

*[PDF version](https://berkeleygenomics.org/pdfs/Chromosome_identification_methods.pdf). [berkeleygenomics.org](https://berkeleygenomics.org/articles/Chromosome_identification_methods.html). [x.com](https://x.com/BerkeleyGenomic/status/2005883397680357476). [bluesky](https://bsky.app/profile/berkeleygenomics.bsky.social/post/3mb6p6swuk225).*


*This is a linkpost for 
"[Chromosome identification methods](https://berkeleygenomics.org/articles/Chromosome_identification_methods.html)"; a few of the initial sections are reproduced here.*

# Abstract

Chromosome selection is a hypothetical technology that assembles the genome of a new living cell out of whole chromosomes taken from multiple source cells. To do chromosome selection, you need a method for chromosome identification—distinguishing between chromosomes by number, and ideally also by allele content. This article investigates methods for chromosome identification. It seems that existing methods are subject to a tradeoff where they either destroy or damage the chromosomes they measure, or else they fail to confidently identify chromosomes. A paradigm for non-destructive high-confidence chromosome identification is proposed, based on the idea of complementary identification. The idea is to isolate a single chromosome taken from a single cell, destructively identify all the remaining chromosomes from that cell, and thus infer the identity of the preserved chromosome. The overall aim is to eventually develop a non-destructive, low-cost, accurate way to identify single chromosomes, to apply as part of a chromosome selection protocol.

# Context

Reprogenetics is biotechnology to empower parents to make genomic choices on behalf of their future children. One key operation that's needed for reprogenetics is [genomic vectoring](https://berkeleygenomics.org/articles/Methods_for_strong_human_germline_engineering.html#genomic-vectoring-gv): creating a cell with a genome that's been modified in some specific direction. 

Chromosome selection is one possible genomic vectoring method. It [could be fairly powerful](https://berkeleygenomics.org/articles/Methods_for_strong_human_germline_engineering.html#method-chromosome-selection) if applied to sperm chromosomes or applied to multiple donors. The basic idea is to take several starting cells, select one or more chromosomes from each of those cells, and then put all those chromosomes together into one new cell:

![](https://i.imgur.com/kDh3ZCw.png)

There are three fundamental elements needed to perform chromosome selection:

* *Transmission* and *Exclusion*. Get some chromosomes into the final cell, while excluding some other chromosomes.

* *Targeting*. Differentially apply transmission and exclusion to different chromosomes.

This article deals with the *targeting* element. Future articles will deal with the other elements. Specifically, this article tries to answer the question: 

> How can we *identify* chromosomes?

That is, how can we come to know the *number* of one or more chromosomes that we are handling (i.e. is it chromosome 1, or chromosome 2, etc.)? Further, how can we come to know what alleles are contained in the specific chromosome we are handling, among whatever alleles are present among the chromosomes we're selecting from?

This problem has been approached from many angles. There are several central staples of molecular biology, such as DNA sequencing, karyotyping, flow cytometry, CRISPR-Cas9, and FISH; and there are several speculative attempts to study chromosomes in unusual ways, such as acoustics, laser scattering, hydrodynamic sorting, and electrokinesis. 

This article presents an attempt to sort through these methods and find ones that will work well as part of a chromosome selection method. This goal induces various constraints on methods for chromosome identification; hopefully future articles will further clarify those constraints. 

# Synopsis and takeaways


A human cell has 46 chromosomes, 2 of each number, with each number (and X and Y) being of different sizes:

![](https://i.imgur.com/gSlc7CA.png)

*[([Figure 1.3](https://bookdown.org/maria_gallegos/where-are-genes-2021/genes-genomes-and-genome-browsers.html) from Gallegos (2022)[^gallegos]. © publisher)]*

[^gallegos]: Gallegos, Maria. Fantastic Genes and Where to Find Them. Updated 2022-09-13. Accessed 16 February 2025. <https://bookdown.org/maria_gallegos/where-are-genes-2021/#preface>.

We want to identify chromosomes. Technically, that means we want to be able to somehow operate differently on chromosomes of different numbers. In practice, for the most part, what we want is to *isolate* one or more chromosomes, and then *learn* what number(s) they are. (If possible, we also want to learn what alleles they carry.)

How do we identify chromosomes? We have to measure them somehow. 

There's a tradeoff between different ways of measuring chromosomes: How much *access* do you have to the DNA inside the chromosome? (Chromosomes are not just DNA; they also incorporate many proteins.)

On one extreme, there is, for example, standard DNA sequencing. In this method, you have lots of direct access to the DNA, so you can easily measure it with very high confidence, and learn the number of a chromosome and almost all of the alleles it carries. However, this method is also completely destructive. You strip away all the proteins from the DNA, you disrupt the epigenetic state of the DNA, and you chop up the DNA into tiny little fragments. High DNA access comes with high information, but also comes with high destructiveness.

On the other extreme, there is, for example, standard light microscopy. In this method, you have very little direct access to the chromosome's DNA. You just shine light on the chromosome and see what you can see. This method is not at all destructive; the chromosome's DNA, structural proteins, and epigenetic state are all left perfectly intact. However, this method definitely cannot tell you what alleles the chromosome carries, and may not even be able to distinguish many chromosomes by number. Low DNA access comes with low destructiveness, but also comes with low information.

If we're assembling a new cell (for example, to use in place of a sperm), we cannot use chromosomes that we have destroyed. We also (roughly speaking) cannot use a chromosome unless we're confident we know what number it is, because we have to be confident that the final cell will be euploid. Are there methods that are non-destructive and also make confident calls about chromosome number? 

I don't know of a theoretical reason such a method should not exist. Why not measure physical properties of a chromosome from a distance and infer its number? For example, a single paper from 2006 claimed to use Raman spectroscopy to distinguish with fairly high confidence between human chromosomes 1, 2, and 3, just by bouncing (scattering) a laser off of them[^ojeda_raman]. However, all such methods I've looked at are similar, in that they are very poorly refined: they have not been extensively replicated, so they may not work at all, and definitely have not been developed to be easy and reliable. 

Therefore, as far as I know, there is currently probably no good way to identify chromosomes by directly measuring them. Every single such method will destroy the chromosome, or will not make confident calls about the chromosome's number, or else has not been well-demonstrated to work. Here's a visual summary of the situation:

![](https://i.imgur.com/2pKjHdQ.png)

*[(Hi r/ChartCrimes!)]*

Sidenote: Many readers might wonder: Why not just use standard cell culture sequencing? The reason will be explained more fully in a future article. But basically, the reason is that ensembling a target genome using cell culturing methods (such as [MMCT](https://en.wikipedia.org/wiki/Microcell-mediated_chromosome_transfer)) is likely to be very inconvenient. To avoid that, we want a more reductive mechanical method, an "[isolating-ensembling](#cell-culturing-vs.-isolating-ensembling-methods)" method, where we isolate single chromosomes, identify them, and then put target chromosomes into a new cell. Isolating-ensembling methods require a way to identify single chromosomes (or small sets of chromosomes); it's not enough to just learn the content of some full euploid genomes, which is all that is offered by cell culture sequencing.


So, if we cannot identify chromosomes by directly measuring them, what to do? 

My proposal is to identify chromosomes by *indirectly* measuring them. To indirectly measure a chromosome, we get some material that comes from the same place as the chromosome. We then directly measure that material, and use that measurement to infer something about the chromosome:

![](https://i.imgur.com/JpiK6FB.png)

A key indirect identification method is complementary chromosome identification. That's where you take a single cell with a known genome, isolate one chromosome, and then sequence the rest of the chromosomes. This tells you the identity of the isolated chromosome, without ever directly measuring that chromosome:

![](https://i.imgur.com/fTKFQUG.png)

(See the subsection "[Chromosome-wise complementary identification](#chromosome-wise-complementary-identification)".)


Another indirect identification method is single-cell RNA sequencing for sperm. This works by separating out RNAs from a single sperm and sequencing them. It turns out that those RNAs actually tell you which *alleles* are present in that sperm's genome. (See the subsection "[Sequencing post-meiotic RNA](#sequencing-post-meiotic-rna)".) This tells you the *set* of chromosomes you have, including what crossovers happened. (Another way to do this might be to briefly culture the sperm as haploid cells using donor oocytes[^Metacelsus_2025]; see the subsection "[Haploid culture](#haploid-culture)".)




By combining complementary chromosome number identification with one of these indirect allele-measuring methods ("setwise homolog identification"), we could in theory isolate a single fully intact chromosome with a confidently, almost completely known genome. 

![](https://i.imgur.com/nNOkTU0.png)

This would be a good solution to chromosome identification. Unfortunately, these methods would be very challenging to actually develop. But, that effort might be worth it, since it seems there are not better chromosome identification methods available. See future articles for discussion of how to implement these methods.

The rest of this article will go into much more detail on many of the above points.

[^Metacelsus_2025]: Metacelsus. ‘Androgenetic Haploid Selection’. Substack newsletter. De Novo, 16 November 2025. <https://denovo.substack.com/p/androgenetic-haploid-selection>.

[^ojeda_raman]: Ojeda, Jenifer F., Changan Xie, Yong-Qing Li, Fred E. Bertrand, John Wiley, and Thomas J. McConnell. ‘Chromosomal Analysis and Identification Based on Optical Tweezers and Raman Spectroscopy’. Optics Express 14, no. 12 (2006): 5385–93. <https://doi.org/10.1364/OE.14.005385>