{"id":2792,"date":"2016-01-11T18:01:55","date_gmt":"2016-01-11T18:01:55","guid":{"rendered":"http:\/\/www.blopig.com\/blog\/?p=2792"},"modified":"2016-01-11T18:01:55","modified_gmt":"2016-01-11T18:01:55","slug":"next-generation-sequencing-of-paired-heavy-and-light-chain-sequences","status":"publish","type":"post","link":"https:\/\/www.blopig.com\/blog\/2016\/01\/next-generation-sequencing-of-paired-heavy-and-light-chain-sequences\/","title":{"rendered":"Next generation sequencing of paired heavy and light chain sequences"},"content":{"rendered":"

At the last meeting before Christmas I covered the article <\/a>by DeKosky et al.<\/em> describing a new methodology for sequencing of paired VH-VL repertoire<\/strong> developed by the authors.<\/p>\n

In the recent years there have been an exponential growth of available antibody sequences<\/strong>, caused mainly by the development of cheap and high-throughput Next Generation Sequencing<\/strong> (NGS) technologies. This trend led to the creation of several publicly available antibody sequence databases<\/strong> such as the DIGIT<\/a> database and the abYsis<\/a> 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<\/strong>, with the available techniques<\/a> suffering from low throughput (<700 cells) and high cost. In contrast, the method developed by DeKosky et al.<\/em> allows for relatively cheap paired sequencing of most of the 10^6 B cells contained within a typical 10-ml blood draw.<\/p>\n

The work flow<\/strong> 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<\/strong>. Next, the cell lysis<\/strong> occurs within the droplets and the mRNA<\/strong> fragments coding for the antibody chains attach to the poly(dT) beads<\/strong>. Following that, the mRNA fragments are recovered and linkage PCR is used to generate 850 bp cDNA fragments for NGS<\/strong>.<\/p>\n

To analyse the accuracy of their methodology the authors sequenced paired CDR-H3 \u2013 CDR-L3 sequences<\/strong> from blood samples obtained from three different human donors, filtering the results by 96% clustering, read-quality<\/strong> and removing sequences with less than two reads<\/strong>. Overall, this resulted in ~200,000<\/strong> paired CDR-H3 \u2013 CDR-L3 sequences. The authors found that pairing accuracy of their methodology was ~98%.<\/strong><\/p>\n

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 \u201cpromiscuous<\/strong>\u201d CDR-L3s are also \u201cpublic<\/strong>\u201d 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<\/strong>. The results also show that the promiscuous CDR-L3s show little to no modification<\/strong>, being very close to the germline sequence<\/strong>.<\/p>\n

\"\"<\/a>

Illustration of the sequencing pipeline<\/p><\/div>\n

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

Finally, the authors looked at the occurrence of traits commonly associated with broadly Neutralizing Antibodies (bNAbs)<\/strong>, produced to fight rapidly mutating pathogens (such as the influenza virus). These traits were short (<6 aa) CDR-L3 and long (<\/strong>11 \u2013 18 aa<\/strong>) CDR-H3s<\/strong>. In total, the authors found 31 sequences with these features<\/strong>, suggesting that bNAbs can be found in the repertoire of healthy donors.<\/p>\n

Overall this article presents very interesting and promising method<\/strong>, that should allow for large-scale sequencing of paired VH-VL sequences.<\/p>\n","protected":false},"excerpt":{"rendered":"

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) […]<\/p>\n","protected":false},"author":31,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"nf_dc_page":"","wikipediapreview_detectlinks":true,"_monsterinsights_skip_tracking":false,"_monsterinsights_sitenote_active":false,"_monsterinsights_sitenote_note":"","_monsterinsights_sitenote_category":0,"ngg_post_thumbnail":0,"_jetpack_memberships_contains_paid_content":false,"footnotes":""},"categories":[10],"tags":[],"ppma_author":[493],"class_list":["post-2792","post","type-post","status-publish","format-standard","hentry","category-groupmeetings"],"jetpack_featured_media_url":"","jetpack_sharing_enabled":true,"authors":[{"term_id":493,"user_id":31,"is_guest":0,"slug":"jaroslaw","display_name":"jaroslaw nowak","avatar_url":"https:\/\/secure.gravatar.com\/avatar\/d3ddaccc000bffff018a1bdedaebefb1ac9454506ae5203150e7b3efd8dd9a6e?s=96&d=mm&r=g","0":null,"1":"","2":"","3":"","4":"","5":"","6":"","7":"","8":""}],"_links":{"self":[{"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/posts\/2792","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/users\/31"}],"replies":[{"embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/comments?post=2792"}],"version-history":[{"count":4,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/posts\/2792\/revisions"}],"predecessor-version":[{"id":2797,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/posts\/2792\/revisions\/2797"}],"wp:attachment":[{"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/media?parent=2792"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/categories?post=2792"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/tags?post=2792"},{"taxonomy":"author","embeddable":true,"href":"https:\/\/www.blopig.com\/blog\/wp-json\/wp\/v2\/ppma_author?post=2792"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}