Next generation sequencing of paired heavy and light chain sequences

At the last meeting before Christmas I covered the article by DeKosky et al. describing a new methodology for sequencing of paired VH-VL repertoire developed by the authors.

In the recent years there have been an exponential growth of available antibody sequences, caused mainly by the development of cheap and high-throughput Next Generation Sequencing (NGS) technologies. This trend led to the creation of several publicly available antibody sequence databases such as the DIGIT database and the abYsis database, containing hundreds of thousands of unpaired light chain and heavy chain sequences from over 100 species. Nevertheless, the sequencing of paired VH-VL repertoire remained a challenge, with the available techniques suffering from low throughput (<700 cells) and high cost. In contrast, the method developed by DeKosky et al. allows for relatively cheap paired sequencing of most of the 10^6 B cells contained within a typical 10-ml blood draw.

The work flow is as follows: first the isolated cells, dissolved in water, and magnetic poly(dT) beads mixed with cell lysis buffer are pushed through a narrow opening into a rapidly moving annular oil phase, resulting in a thin jet that coalescences into droplets, in such a way that each droplet has a very low chance of having a cell inside it. This ensures that the vast majority of droplets that do contain cells, contain only one cell each. Next, the cell lysis occurs within the droplets and the mRNA fragments coding for the antibody chains attach to the poly(dT) beads. Following that, the mRNA fragments are recovered and linkage PCR is used to generate 850 bp cDNA fragments for NGS.

To analyse the accuracy of their methodology the authors sequenced paired CDR-H3 – CDR-L3 sequences from blood samples obtained from three different human donors, filtering the results by 96% clustering, read-quality and removing sequences with less than two reads. Overall, this resulted in ~200,000 paired CDR-H3 – CDR-L3 sequences. The authors found that pairing accuracy of their methodology was ~98%.

The article also contained some bioinformatics analysis of the data. The authors first analysed CDR-L3 sequences that tend to pair up with many diverse CDR-H3 sequences and whether such “promiscuous” CDR-L3s are also “public” i.e. they are promiscuous and common in all three donors. Their results show that out of 50 most common promiscuous CDR-L3s 49 are also public. The results also show that the promiscuous CDR-L3s show little to no modification, being very close to the germline sequence.

Illustration of the sequencing pipeline

The sequencing data also contained examples of allelic inclusion, where one B-cell expresses two B cell receptors (almost always one VH gene and two distinct VL genes). It was found that about ~0.5% of all analysed B-cells showed allelic inclusion.

Finally, the authors looked at the occurrence of traits commonly associated with broadly Neutralizing Antibodies (bNAbs), produced to fight rapidly mutating pathogens (such as the influenza virus). These traits were short (<6 aa) CDR-L3 and long (11 – 18 aa) CDR-H3s. In total, the authors found 31 sequences with these features, suggesting that bNAbs can be found in the repertoire of healthy donors.

Overall this article presents very interesting and promising method, that should allow for large-scale sequencing of paired VH-VL sequences.

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