Commit 3f599527 authored by Mathieu Giraud's avatar Mathieu Giraud

Merge branch 'doc/library-preparation-sequencing' into 'dev'

Some documentation on library preparation and sequencing

See merge request !568
parents ee1c8ac6 2bcf49f3
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sequencing data from lymphocytes.** [V(D)J recombinations](http://en.wikipedia.org/wiki/V\(D\)J_recombination) in lymphocytes are
essential for immunological diversity. They are also useful markers of
pathologies, and in leukemia, are used to quantify the minimal residual
disease during patient follow-up. High-throughput sequencing (NGS/HTS) now
disease during patient follow-up.
With adapted [library preparation and sequencing](locus.md),
high-throughput sequencing (NGS/HTS) now
enables the deep sequencing of a lymphoid population with dedicated [Rep-Seq](http://omictools.com/rep-seq-c424-p1.html)
methods and software.
......
# Sequencing and analyzing human immune repertoires
[V(D)J recombinations](http://en.wikipedia.org/wiki/V\(D\)J_recombination) in lymphocytes are essential for immunological diversity.
They are also useful markers of pathologies, and in leukemia, are used to quantify the minimal residual disease during patient follow-up.
High-throughput sequencing (NGS/HTS) now enables the deep sequencing of a lymphoid population with dedicated [RepSeq](http://omictools.com/rep-seq-c424-p1.html) methods and software.
# Library preparation and sequencing for human RepSeq studies
Choosing library preparation and sequencing for immune repertoire analysis
is a challenging task [(Langerak 2017)](http://dx.doi.org/10.4049/jimmunol.1602050)
and depends of multiple factors: aim of the study, people, sequencers, reagents, costs...
We do not aim here to be authoritative,
but give a few links to commonly used strategies for library preparation and sequencing.
## Amplicon-based strategies
PCR approaches are the state-of-the-art way to detect
and quantify immune recombinations.
- As of 2020, it is recommended to use the **EuroClonality-NGS** primer sets
published in [(Brüggemann, 2019)](http://dx.doi.org/10.1038/s41375-019-0496-7)
(2-step, 138 primers in 8 tubes, IGH FR1, IGH+, IGK, IGK+, TRB, TRB+, TRD/TRD+, TRG)
and in [(Scheijen, 2019)](http://dx.doi.org/10.1038/s41375-019-0508-7)
(1-step, 53 primers in 3 tubes, IGH FR3, IGH+, IGK, IGK+).
These primer sets were evaluated in a multi-center validation study.
The EuroClonality-NGS consortium also published
[standard operating procedures](http://www.euroclonality.org/protocols)
for Illumina MiSeq and Ion Torrent, that can be adapted for other sequencers.
<br />
Download: [2019-EuroClonality-NGS-primers.csv](http://www.vidjil.org/data/2019-EuroClonality-NGS-primers.csv)
- Many studies are still successfully using primer sets based on
the older **EuroClonality/BIOMED-2** sets
published in [(van Dongen, 2003)](http://dx.doi.org/10.1038/sj.leu.2403202).
See for example [(Ferret, 2016)](http://dx.doi.org/10.1111/bjh.13981)
(1-step, 23 primers in 5 tubes, TRG, TRD/TRD+, IGK, IGK+).
These primer sets were designed and evaluated for onco-hematological studies on lymphoma and/or leukemia samples
but may also be used in other studies on the immune repertoire.
Such primer sets or DNA-Seq (or even on RNA-Seq) are very specific,
leading to usually datasets with more than 90% or 99% of reads with V(D)J recombinations.
One-step approaches may be used even with the 2-steps primers,
see [(Brüggemann, 2019)](http://dx.doi.org/10.1038/s41375-019-0496-7) for discussion.
Some labs do sequence independently the tubes with barcoding,
but for many applications the contents of the tubes can be pooled and sequenced at once.
Using the full depth of a recent sequencer with spike-in control sequences,
precise MRD quantification can be achieved [(Knecht 2019)](http://dx.doi.org/10.1038/s41375-019-0499-4).
When the goal is only to detect a few dominant clones,
many samples (10 to 100, or even more)
can be pooled with proper barcoding in a same sequencing run.
Contamination should then particularly be monitored.
## Capture and other strategies
Several library preparations on DNA or RNA can be done with limited or no amplification:
whole-genome or whole-transcriptome sequencing, capture, 5'RACE...
Probes can possibly be designed in every V, D, or J gene, in the constant region, and/or
consensus probes can be used.
These methods can also be applied on single-cell sequencing, possibly with UMI identifiers.
One advantage of such libraries is that they can be combined to other studies,
as for example with transcriptome analysis
or probes targeting oncogenes or other sequences of interest.
Of course, the downside is that non-recombined DNA or RNA are also sequenced:
Depending on the method and the datasets,
as few as between 0.001% and 0.1% reads will have an actual V(D)J recombination.
With datasets with billions of reads,
this is usally enough to detect dominant clones
with something like a few hundred reads,
but quantification is more limited.
# Analyzed human immune recombinations in Vidjil
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Vidjil is an open-source platform for the analysis of high-throughput sequencing data from lymphocytes.
[V(D)J recombinations](http://en.wikipedia.org/wiki/V\(D\)J_recombination) in lymphocytes are essential for immunological diversity.
They are also useful markers of pathologies, and in leukemia, are used to quantify the minimal residual disease during patient follow-up.
High-throughput sequencing (NGS/HTS) now enables the deep sequencing of a lymphoid population with dedicated [Rep-Seq](http://omictools.com/rep-seq-c424-p1.html) methods and software.
With adapted [library preparation and sequencing](http://www.vidjil.org/doc/locus),
high-throughput sequencing (NGS/HTS) now enables the deep sequencing of a lymphoid population with dedicated [Rep-Seq](http://omictools.com/rep-seq-c424-p1.html) methods and software.
This is the help of the [Vidjil web application](http://app.vidjil.org/).
Further help can always be asked to <support@vidjil.org>. We can also arrange phone or video meeting.
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......@@ -26,6 +26,8 @@ Other documentation (users and administrators of the web application, developper
diversity. They are also useful markers of pathologies, and in
leukemia, are used to quantify the minimal residual disease during
patient follow-up.
With adapted [library preparation and sequencing](http://www.vidjil.org/doc/locus),
high-throughput sequencing (NGS/HTS) now enables the deep sequencing of a lymphoid population with dedicated [Rep-Seq](http://omictools.com/rep-seq-c424-p1.html) methods and software.
Vidjil-algo processes high-throughput sequencing data to extract V(D)J
junctions and gather them into clones. Vidjil-algo starts
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......@@ -10,7 +10,7 @@ repo_name: gitlab
pages:
- / : index.md
- user manual: user.md
- recombinations: locus.md
- libraries, recombinations: locus.md
- vidjil-algo: vidjil-algo.md
- .vidjil format: vidjil-format.md
- admin: admin.md
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