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So! You wish to search all the general public metagenomes with a genome sequence!


Think about you could have a (microbial) genome. Or a contig. And also you wish to
discover comparable sequences, both in genomes or in metagenomes.

On the lookout for it in genomes is feasible, if not at all times simple – you may go
to NCBI and do a BLAST of some kind, however BLAST is meant for extra
delicate and shorter matches. However there are different instruments, together with
sourmash, a instrument we have been
growing for a number of years, that can fortunately do it for you.

On the lookout for one thing in metagenomes is tougher. Metagenomes are
lots of, 1000’s, and even thousands and thousands of occasions bigger than genomes,
and doing something with them shortly is difficult. sourmash helps
doing it one metagenome at a time, however it’s sluggish and reminiscence intensive;
serratus will do it for you utilizing the ability of
the cloud, however it’ll price you (not less than) a number of thousand $$.

When you’re considering how we’re doing DNA sequence search, this is an
excerpt from
a earlier weblog put up about utilizing SQLite to retailer our information

The fundamental thought is that we take lengthy DNA sequences, extract
sub-sequences of a set size (say okay=31), hash them, after which sketch
them by retaining solely people who fall beneath a sure threshold
worth. Then we seek for matches between sketches based mostly on variety of
overlapping hashes. It is a proxy for the variety of overlapping okay=31
subsequences, which is in flip convertible into varied sequence
similarity metrics.

MAGsearch exists! It really works! Nevertheless it’s exhausting to share.

For a few years now, we have had one thing known as
MAGsearch working
on our personal non-public infrastructure. MAGsearch is sourmash on steroids:
it makes use of the identical underlying Rust library as sourmash and masses and
searches the metagenomes shortly. And it’ll do all of this on
commodity {hardware} that many individuals have entry to – a search of as much as
a thousand genomes towards the SRA takes beneath 12 GB of RAM, and beneath
11 hours, utilizing 32 cores.

MAGsearch does a reasonably easy factor: it masses all of the question
genomes into reminiscence after which iteratively masses every of ~700,000
metagenome sketches, reporting any overlaps. It does so in parallel,
which is why it is so quick – doing this with sourmash would take about
40 occasions as lengthy, as a result of sourmash is not parallelized.

One drawback with MAGsearch is that it isn’t actual time. 10 hours is
nice!!, particularly for 1000 genomes, however that is nonetheless solely about two
genomes a minute. And it is too sluggish for us to offer MAGsearch as a
service.

One other drawback is that the underlying information is about 10 TB on the
second, and we do not actually have a method to share that information.

So we have been utilizing MAGsearch a good bit during the last two years to do
searches for others, however it’s at all times completed in a type of batch mode
the place we run it in between different issues we’re doing.

Enter ‘mastiff’ – utilizing RocksDB to do issues quicker

For the
2022 JGI Person Assembly
Dr. Luiz Irber was invited to speak about his MAGsearch work, and he
acquired impressed to check out another answer.

He determined to implement an inverted index utilizing
RocksDB, an embeddable database. I have not dug
into the implementation,
however I imagine mastiff makes use of particular person hashes as keys and shops a
vector of dataset IDs as values. So a seek for overlaps within the
database is completed by utilizing hashes from a question as keys, after which
intersecting the hashes within the values to seek out which dataset IDs have
enough estimated overlap to be reported.

Luiz reported that it took a bit beneath three weeks to construct a RocksDB
index for 500,000 datasets at okay=21, scaled=1000. The ensuing
database is about 700 GB. He then wrote a Net server to allow queries
towards the database.

mastiff permits real-time search of SRA-scale information units!

So… it is quick. Like, actually quick.

It is so quick, you may simply go strive it out your self – I’ve supplied up a
easy pocket book
right here
in
this github repo,
and you’ll run it instantly by clicking on the button beneath:

This pocket book does the next:

  • downloads some SRA metadata (as soon as)
  • masses and sketches a Shewanella genome question right into a sourmash signature (~45 KB, for a ~5.3 Mbp genome)
  • serializes the signatures and sends it to the mastiff server to run it towards the SRA
  • receives the ensuing CSV of dataset + containment estimates
  • interprets the CSV in gentle of the SRA metadata

What you may see on the backside of the pocket book is that this specific
genome tends to point out up in freshwater and wastewater.

The cool factor is you can run your individual queries when you like – simply
exchange the shewanella.fa.gz file references with your individual queries
of curiosity!

(There’s additionally
a snakemake workflow to question
mastiff if you wish to run many queries, and a mastiff command-line
program that can sketch and question multi function go.)

What can mastiff be used for?

MAGsearch is already being utilized by individuals for
outbreak evaluation
and biogeography research, amongst different issues. We now have a number of completely different
lively analysis tasks within the lab which can be exploring its utility for
varied questions. So we are going to quickly have the ability to do these issues so much
quicker. Yay!

I personally am trying ahead to digging into pressure dynamics and
content-based alerts of latest metagenomes, amongst different issues.

We will additionally allow different cool tasks, together with (maybe most
importantly) issues that we did not consider.

A rule of thumb that I like is {that a} expertise will likely be most helpful
for researchers when a summer season undergrad can casually use it to discover
wild-haired concepts and provoke summer season tasks based mostly on quickly
generated exploratory outcomes – and I am actually curious to see what we
can allow others to do with this ;). I can think about that after individuals
can casually search the SRA with queries, they will provide you with numerous
concepts and make numerous discoveries. (In fact, numerous follow-up work
could be wanted, too – chasing down what detection of a genome in a
metagenome means biologically is hard!)

It has not escaped our discover that this can be utilized for a lot smaller
databases, too. So we’re trying ahead to enabling real-time search
of all of the NCBI microbial genomes, in addition to ..nicely, no matter we are able to
get our arms on :).

mastiff will ultimately (see beneath, “Whither mastiff?”) be built-in
into sourmash and/or robustified, after which it’ll help non-public
databases, too.

Effectively, however wait, you mentioned “real-time”

Proper, I did – it takes between 2 and 10 seconds to do a search, and
IIRC the server can deal with as much as 200 simultaneous queries at a time.

And I’ve gotta be sincere… at first I missed the purpose that this was
real-time. And web-enabled.

I used to be describing it to some collaborators, and whereas I used to be describing
it I spotted, oh, cool, we are able to truly do that all in JavaScript by way of
WebAssembly too, after all.

So, additionally coming ultimately (if not, like, tomorrow), I count on we are going to
present a Site the place you may sketch a genome client-side (e.g. in
the browser – see
sourmash#1973),
after which obtain near-instantaneous reporting on similarities to any
recognized genome in addition to presence inside public metagenomes.

And, as soon as varied issues are labored out , I hope we are able to present
this as a generic service for others to make use of.

In order that appears neat, proper?

Cautions, reservations, and limitations

There are some things you need to know earlier than you get too excited. I
imply, you need to completely be excited, however… learn on.

First, this can be a proof of idea. It reveals it may be completed, however it’s
not (but) one thing that anybody aside from Luiz can run! Engineering
and testing and releasing must occur, and that can take time.

Second, there are fairly important limitations to this on the
scientific aspect. The search will solely work out to about
90% common nucleotide identification (ANI) – a containment of .01-.05,
which implies you may robustly discover matches out to the genus stage, however
not past. That is a limitation of nucleotide k-mers and it is
one thing we’re engaged on.

Small-ish queries additionally do not work nicely – we are able to robustly discover actual
matches to 10kb chunks of sequence, however not shorter.

Third, mastiff is generally designed round looking for small
queries. Question occasions ought to scale roughly linearly with the
question dimension. Luiz has restricted the server to a 5MB question for this
cause.

And final however in no way least, that is not the whole SRA, it is
solely about 480,000 information (of about 700,000). We’ll replace it
ultimately, however for now it is a enough proof of idea ;).

Whither mastiff?

We (largely Luiz 😉 are working to combine mastiff performance into
sourmash. There is a fairly large hole between a proof-of-concept
implementation and mature, sturdy, end-user-usable software program, of
course, however we all know methods to do it.

There’s most likely different tremendous cool back-end approaches we may use,
and we would love to speak to you about them when you’re considering attempting
out various implementations. At this level we have now a reasonably good
understanding of the conceptual operations and may even convey them to
you in functioning code snippets :).

I additionally gotta let you know that we do not know methods to help this type of
work precisely. This developed out of Luiz’s thesis work however is now completed
on a volunteer foundation by him. JGI is supporting the server improvement
for a yr (thanks!!) however we’re a bit bottlenecked on UX help and
backend/frontend improvement. So
drop us a line if
you have acquired some spare change – we would be searching for 3-5 years of
help.

(I would be considering exploring governance and sustainability points
round this type of factor, too.)

Acknowledgements

The interpretation and understanding of MAGsearch outcomes has been
tremendously helped by work from Dr. Tessa Pierce-Ward (ANI),
Dr. Adrian Viehweger (pathogen outbreaks), Dr. Jessica Lumian
(biogeography), Dr. Christy Grettenberger (biogeography and extra), and
others. Thanks!!



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