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Tagging systems for sequencing large cohorts
KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Gene Technology. (Gene Technology)
2010 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Advances in sequencing technologies constantly improves the throughput andaccuracy of sequencing instruments. Together with this development comes newdemands and opportunities to fully take advantage of the massive amounts of dataproduced within a sequence run. One way of doing this is by analyzing a large set ofsamples in parallel by pooling them together prior to sequencing and associating thereads to the corresponding samples using DNA sequence tags. Amplicon sequencingis a common application for this technique, enabling ultra deep sequencing andidentification of rare allelic variants. However, a common problem for ampliconsequencing projects is formation of unspecific PCR products and primer dimersoccupying large portions of the data sets.

This thesis is based on two papers exploring these new kinds of possibilities andissues. In the first paper, a method for including thousands of samples in the samesequencing run without dramatically increasing the cost or sample handlingcomplexity is presented. The second paper presents how the amount of high qualitydata from an amplicon sequencing run can be maximized.

The findings from the first paper shows that a two-tagging system, where the first tagis introduced by PCR and the second tag is introduced by ligation, can be used foreffectively sequence a cohort of 3500 samples using the 454 GS FLX Titaniumchemistry. The tagging procedure allows for simple and easy scalable samplehandling during sequence library preparation. The first PCR introduced tags, that arepresent in both ends of the fragments, enables detection of chimeric formation andhence, avoiding false typing in the data set.

In the second paper, a FACS-machine is used to sort and enrich target DNA covered emPCR beads. This is facilitated by tagging quality beads using hybridization of afluorescently labeled target specific DNA probe prior to sorting. The system wasevaluated by sequencing two amplicon libraries, one FACS sorted and one standardenriched, on the 454 showing a three-fold increase of quality data obtained.

Place, publisher, year, edition, pages
Stockholm , 2010. , 38 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2010:15
Keyword [en]
next generation sequencing, genotyping, massive parallel sequencing, 454, Pyrosequencing, amplicon sequencing, enrichment, DNA barcodes
National Category
Genetics
Identifiers
URN: urn:nbn:se:kth:diva-24365ISBN: 978-91-7415-706-2 (print)OAI: oai:DiVA.org:kth-24365DiVA: diva2:349196
Presentation
2010-09-24, FA32, Roslagstullsbacken 21, Stockholm, AlbaNova, 10:15 (Swedish)
Supervisors
Note
QC20100907Available from: 2010-09-07 Created: 2010-09-06 Last updated: 2012-03-23Bibliographically approved
List of papers
1. Decoding a substantial set of samples in parallel by massive sequencing
Open this publication in new window or tab >>Decoding a substantial set of samples in parallel by massive sequencing
2011 (English)In: Plos One, ISSN 1932-6203, Vol. 6, no 3Article in journal (Refereed) Published
Abstract [en]

The dramatic increase of throughput seen in the eld of sequenceanalysis during the last years has opened up new possibilities of se-quencing a multitude of samples in parallel. Here we present a novelstrategy where the combination of two tags is used to link reads totheir origins in a pool of samples. The two tags are incorporated intwo steps leading to lowering of sample handling complexity by nearly100 times. The method described here enables accurate identi cationand typing of thousands of samples in parallel and is scalable. In thisstudy the system was designed to test 4992 samples using only 122 tags.

To proof the concept of two tagging method the highly polymor-phic 2nd exon of DLA-DRB1 in dogs and wolves was sequenced usingthe 454 GS FLX Titanium Chemistry. By requiring a minimum se-quence depth of 20 reads per sample, 94% of the successfully ampli edsamples were genotyped. In addition, the method allowed digital de-tection of chimeric fragments. These results demonstrate that it ispossible to sequence thousands of samples in parallel without complexpooling patterns or primer combinations. Furthermore, the method isscalable and increasing the sample size by 960 samples requires only10 additional tags.

Place, publisher, year, edition, pages
San Fransisco: PUBLIC LIBRARY SCIENCE, 2011
Keyword
HIGH-THROUGHPUT; HIGH-RESOLUTION; DNA; PROBES
National Category
Genetics
Identifiers
urn:nbn:se:kth:diva-24353 (URN)10.1371/journal.pone.0017785 (DOI)000288170900047 ()2-s2.0-79952430921 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20100906 Uppdaterad från manuskript till artikel i tidskrift 20110407

Available from: 2010-09-06 Created: 2010-09-03 Last updated: 2012-11-16Bibliographically approved
2. Gene-specific FACS sorting method for target selection in high-throughput amplicon sequencing
Open this publication in new window or tab >>Gene-specific FACS sorting method for target selection in high-throughput amplicon sequencing
2010 (English)In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 11, no 140Article in journal (Refereed) Published
Abstract [en]

Background

In addition to shotgun sequencing, next generation sequencing has been shown to be suitable for deep sequencing of many specific PCR-amplified target genes in parallel. However, unspecific product formation is a common problem in amplicon sequencing since these fragments are difficult to fully remove by gel purification, and their presence inevitably reduces the number of mappable sequence reads that can be obtained in each sequencing run.

Results

We have used a novel flow cytometric sorting approach to specifically enrich Roche/454 DNA Capture beads carrying target DNA sequences on their surface, and reject beads carrying unspecific sequences. This procedure gives a nearly three-fold increase in the fraction of informative sequences obtained. Presented results also show that there are no significant differences in the distribution or presence of different genotypes between a FACS-enriched sample and a standard-enriched control sample.

Conclusions

Target-specific FACS enrichment prior to Roche/454 sequencing provides a quick, inexpensive way of increasing the amount of high quality data obtained in a single sequencing run, without introducing any sequence bias.

Keyword
sequencing, emPCR, FACS
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-24357 (URN)10.1186/1471-2164-11-140 (DOI)000275835300001 ()2-s2.0-77949406720 (Scopus ID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note
QC 20100906Available from: 2010-09-06 Created: 2010-09-03 Last updated: 2017-12-12Bibliographically approved

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