scholarly journals Novel High-Rank Phylogenetic Lineages within a Sulfur Spring (Zodletone Spring, Oklahoma), Revealed Using a Combined Pyrosequencing-Sanger Approach

2012 ◽  
Vol 78 (8) ◽  
pp. 2677-2688 ◽  
Author(s):  
Noha Youssef ◽  
Brandi L. Steidley ◽  
Mostafa S. Elshahed
Keyword(s):  
High Throughput ◽  
Accurate Determination ◽  
Full Length ◽  
Read Length ◽  
Rrna Gene ◽  
Data Set ◽  
Content Type ◽  
Rare Biosphere ◽  
Phylogenetic Affiliation ◽  
Phylogenetic Lineages

ABSTRACTThe utilization of high-throughput sequencing technologies in 16S rRNA gene-based diversity surveys has indicated that within most ecosystems, a significant fraction of the community could not be assigned to known microbial phyla. Accurate determination of the phylogenetic affiliation of such sequences is difficult due to the short-read-length output of currently available high-throughput technologies. This fraction could harbor multiple novel phylogenetic lineages that have so far escaped detection. Here we describe our efforts in accurate assessment of the novelty and phylogenetic affiliation of selected unclassified lineages within a pyrosequencing data set generated from source sediments of Zodletone Spring, a sulfide- and sulfur-rich spring in southwestern Oklahoma. Lineage-specific forward primers were designed for 78 putatively novel lineages identified within the pyrosequencing data set, and representative nearly full-length small-subunit (SSU) rRNA gene sequences were obtained by pairing those primers with reverse universal bacterial primers. Of the 78 lineages tested, amplifiable products were obtained for 52, 32 of which had at least one nearly full-length sequence that was representative of the lineage targeted. Analysis of phylogenetic affiliation of the obtained Sanger sequences identified 5 novel candidate phyla and 10 novel candidate classes (withinFibrobacteres,Planctomycetes, and candidate phyla BRC1, GN12, TM6, TM7, LD1, WS2, and GN06) in the data set, in addition to multiple novel orders and families. The discovery of multiple novel phyla within a pilot study of a single ecosystem clearly shows the potential of the approach in identifying novel diversities within the rare biosphere.

scholarly journals A method for high precision sequencing of near full-length 16S rRNA genes on an Illumina MiSeq

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2492 ◽  
Author(s):  
Catherine M. Burke ◽  
Aaron E. Darling
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Single Molecule ◽  
Illumina Miseq ◽  
Full Length ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Bacterial Taxonomy

BackgroundThe bacterial 16S rRNA gene has historically been used in defining bacterial taxonomy and phylogeny. However, there are currently no high-throughput methods to sequence full-length 16S rRNA genes present in a sample with precision.ResultsWe describe a method for sequencing near full-length 16S rRNA gene amplicons using the high throughput Illumina MiSeq platform and test it using DNA from human skin swab samples. Proof of principle of the approach is demonstrated, with the generation of 1,604 sequences greater than 1,300 nt from a single Nano MiSeq run, with accuracy estimated to be 100-fold higher than standard Illumina reads. The reads were chimera filtered using information from a single molecule dual tagging scheme that boosts the signal available for chimera detection.ConclusionsThis method could be scaled up to generate many thousands of sequences per MiSeq run and could be applied to other sequencing platforms. This has great potential for populating databases with high quality, near full-length 16S rRNA gene sequences from under-represented taxa and environments and facilitates analyses of microbial communities at higher resolution.

scholarly journals Phylogeny of Intestinal Ciliates, Including Charonina ventriculi, and Comparison of Microscopy and 18S rRNA Gene Pyrosequencing for Rumen Ciliate Community Structure Analysis

2015 ◽  
Vol 81 (7) ◽  
pp. 2433-2444 ◽  
Author(s):  
Sandra Kittelmann ◽  
Savannah R. Devente ◽  
Michelle R. Kirk ◽  
Henning Seedorf ◽  
Burk A. Dehority ◽  
...  
Keyword(s):  
Community Structure ◽  
High Throughput ◽  
18S Rrna ◽  
Gene Sequence ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Content Type ◽  
Advantages And Disadvantages ◽  
Ciliate Community

ABSTRACTThe development of high-throughput methods, such as the construction of 18S rRNA gene clone or pyrosequencing libraries, has allowed evaluation of ciliate community composition in hundreds of samples from the rumen and other intestinal habitats. However, several genera of mammalian intestinal ciliates have been described based only on morphological features and, to date, have not been identified using molecular methods. Here, we isolated single cells of one of the smallest but widely distributed intestinal ciliates,Charonina ventriculi, and sequenced its 18S rRNA gene. We verified the sequence in a full-cycle rRNA approach using fluorescencein situhybridization and thereby assigned an 18S rRNA gene sequence to this species previously known only by its morphology. Based on its full-length 18S rRNA gene sequence,Charonina ventriculiwas positioned within the phylogeny of intestinal ciliates in the subclass Trichostomatia. The taxonomic framework derived from this phylogeny was used for taxonomic assignment of trichostome ciliate 18S rRNA gene sequence data stemming from high-throughput amplicon pyrosequencing of rumen-derived DNA samples. The 18S rRNA gene-based ciliate community structure was compared to that obtained from microscopic counts using the same samples. Both methods allowed identification of dominant members of the ciliate communities and classification of the rumen ciliate community into one of the types first described by Eadie in 1962. Notably, each method is associated with advantages and disadvantages. Microscopy is a highly accurate method for evaluation of total numbers or relative abundances of different ciliate genera in a sample, while 18S rRNA gene pyrosequencing represents a valuable alternative for comparison of ciliate community structure in a large number of samples from different animals or treatment groups.

scholarly journals High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution

2019 ◽  
Vol 47 (18) ◽  
pp. e103-e103 ◽  
Author(s):  
Benjamin J Callahan ◽  
Joan Wong ◽  
Cheryl Heiner ◽  
Steve Oh ◽  
Casey M Theriot ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Amplicon Sequencing ◽  
Full Length ◽  
Rrna Gene ◽  
Single Nucleotide ◽  
Full Complement ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

AbstractTargeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.

scholarly journals Novelty and Uniqueness Patterns of Rare Members of the Soil Biosphere

2008 ◽  
Vol 74 (17) ◽  
pp. 5422-5428 ◽  
Author(s):  
Mostafa S. Elshahed ◽  
Noha H. Youssef ◽  
Anne M. Spain ◽  
Cody Sheik ◽  
Fares Z. Najar ◽  
...  
Keyword(s):  
Phylogenetic Analyses ◽  
Bacterial Species ◽  
Rrna Gene ◽  
Tall Grass ◽  
Data Set ◽  
Bacterial Phyla ◽  
Rare Biosphere ◽  
Soil Bacterial ◽  
Close Relatives ◽  
Taxonomic Groups

ABSTRACT Soil bacterial communities typically exhibit a distribution pattern in which most bacterial species are present in low abundance. Due to the relatively small size of most culture-independent sequencing surveys, a detailed phylogenetic analysis of rare members of the community is lacking. To gain access to the rarely sampled soil biosphere, we analyzed a data set of 13,001 near-full-length 16S rRNA gene clones derived from an undisturbed tall grass prairie soil in central Oklahoma. Rare members of the soil bacterial community (empirically defined at two different abundance cutoffs) represented 18.1 to 37.1% of the total number of clones in the data set and were, on average, less similar to their closest relatives in public databases when compared to more abundant members of the community. Detailed phylogenetic analyses indicated that members of the soil rare biosphere either belonged to novel bacterial lineages (members of five novel bacterial phyla identified in the data set, as well as members of multiple novel lineages within previously described phyla or candidate phyla), to lineages that are prevalent in other environments but rarely encountered in soil, or were close relatives to more abundant taxa in the data set. While a fraction of the rare community was closely related to more abundant taxonomic groups in the data set, a significant portion of the rare biosphere represented evolutionarily distinct lineages at various taxonomic cutoffs. We reason that these novelty and uniqueness patterns provide clues regarding the origins and potential ecological roles of members of the soil's rare biosphere.

scholarly journals Microbial Composition of Human Appendices from Patients following Appendectomy

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Caitriona M. Guinane ◽  
Amany Tadrous ◽  
Fiona Fouhy ◽  
C. Anthony Ryan ◽  
Eugene M. Dempsey ◽  
...  
Keyword(s):  
Human Health ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Evolutionary Development ◽  
Rrna Gene ◽  
Limited Data ◽  
Microbial Composition ◽  
Content Type ◽  
Study Results ◽  
Human Appendix

ABSTRACT The human appendix has historically been considered a vestige of evolutionary development with an unknown function. While limited data are available on the microbial composition of the appendix, it has been postulated that this organ could serve as a microbial reservoir for repopulating the gastrointestinal tract in times of necessity. We aimed to explore the microbial composition of the human appendix, using high-throughput sequencing of the 16S rRNA gene V4 region. Seven patients, 5 to 25 years of age, presenting with symptoms of acute appendicitis were included in this study. Results showed considerable diversity and interindividual variability among the microbial composition of the appendix samples. In general, however, Firmicutes was the dominant phylum, with the majority of additional sequences being assigned at various levels to Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria. Despite the large diversity in the microbiota found within the appendix, however, a few major families and genera were found to comprise the majority of the sequences present. Interestingly, also, certain taxa not generally associated with the human intestine, including the oral pathogens Gemella, Parvimonas, and Fusobacterium, were identified among the appendix samples. The prevalence of genera such as Fusobacterium could also be linked to the severity of inflammation of the organ. We conclude that the human appendix contains a robust and varied microbiota distinct from the microbiotas in other niches within the human microbiome. The microbial composition of the human appendix is subject to extreme variability and comprises a diversity of biota that may play an important, as-yet-unknown role in human health. IMPORTANCE There are currently limited data available on the microbial composition of the human appendix. It has been suggested, however, that it may serve as a “safe house” for commensal bacteria that can reinoculate the gut at need. The present study is the first comprehensive view of the microbial composition of the appendix as determined by high-throughput sequencing. We have determined that the human appendix contains a wealth of microbes, including members of 15 phyla. Important information regarding the associated bacterial diversity of the appendix which will help determine the role, if any, the appendix microbiota has in human health is presented.

scholarly journals Phylogenetic Diversities and Community Structure of Members of the Extremely Halophilic Archaea (Order Halobacteriales) in Multiple Saline Sediment Habitats

2011 ◽  
Vol 78 (5) ◽  
pp. 1332-1344 ◽  
Author(s):  
Noha H. Youssef ◽  
Kristen N. Ashlock-Savage ◽  
Mostafa S. Elshahed
Keyword(s):  
Community Structure ◽  
Salt Lake ◽  
Great Salt Lake ◽  
Halophilic Archaea ◽  
Wide Distribution ◽  
Rrna Gene ◽  
Processing Plant ◽  
Content Type ◽  
Mangrove Tree ◽  
Rare Biosphere

ABSTRACTWe investigated the phylogenetic diversity and community structure of members of the halophilicArchaea(orderHalobacteriales) in five distinct sediment habitats that experience various levels of salinity and salinity fluctuations (sediments from Great Salt Plains and Zodletone Spring in Oklahoma, mangrove tree sediments in Puerto Rico, sediment underneath salt heaps in a salt-processing plant, and sediments from the Great Salt Lake northern arm) usingHalobacteriales-specific 16S rRNA gene primers. Extremely diverseHalobacterialescommunities were encountered in all habitats, with 27 (Zodletone) to 37 (mangrove) different genera identified per sample, out of the currently described 38Halobacterialesgenera. With the exception of Zodletone Spring, where the prevalent geochemical conditions are extremely inhospitable toHalobacterialessurvival, habitats with fluctuating salinity levels were more diverse than permanently saline habitats. Sequences affiliated with the recently described generaHalogranum,Halolamina,Haloplanus,Halosarcina, andHalorientalis, in addition to the generaHalorubrum,Haloferax, andHalobacterium, were among the most abundant and ubiquitous genera, suggesting a wide distribution of these poorly studied genera in saline sediments. TheHalobacterialessediment communities analyzed in this study were more diverse than and completely distinct from communities from typical hypersaline water bodies. Finally, sequences unaffiliated with currently described genera represented a small fraction of the totalHalobacterialescommunities, ranging between 2.5% (Zodletone) to 7.0% (mangrove and Great Salt Lake). However, these novel sequences were characterized by remarkably high levels of alpha and beta diversities, suggesting the presence of an enormous, yet-untapped supply of novelHalobacterialesgenera within the rare biosphere of various saline ecosystems.

scholarly journals Accurate Estimation of Fungal Diversity and Abundance through Improved Lineage-Specific Primers Optimized for Illumina Amplicon Sequencing

2016 ◽  
Vol 82 (24) ◽  
pp. 7217-7226 ◽  
Author(s):  
D. Lee Taylor ◽  
William A. Walters ◽  
Niall J. Lennon ◽  
James Bochicchio ◽  
Andrew Krohn ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Large Subunit ◽  
Marker Gene ◽  
Soil Samples ◽  
Gene Copy ◽  
Accurate Estimation ◽  
Rrna Gene ◽  
Data Set ◽  
Mock Communities

ABSTRACTWhile high-throughput sequencing methods are revolutionizing fungal ecology, recovering accurate estimates of species richness and abundance has proven elusive. We sought to design internal transcribed spacer (ITS) primers and an Illumina protocol that would maximize coverage of the kingdom Fungi while minimizing nontarget eukaryotes. We inspected alignments of the 5.8S and large subunit (LSU) ribosomal genes and evaluated potential primers using PrimerProspector. We tested the resulting primers using tiered-abundance mock communities and five previously characterized soil samples. We recovered operational taxonomic units (OTUs) belonging to all 8 members in both mock communities, despite DNA abundances spanning 3 orders of magnitude. The expected and observed read counts were strongly correlated (r= 0.94 to 0.97). However, several taxa were consistently over- or underrepresented, likely due to variation in rRNA gene copy numbers. The Illumina data resulted in clustering of soil samples identical to that obtained with Sanger sequence clone library data using different primers. Furthermore, the two methods produced distance matrices with a Mantel correlation of 0.92. Nonfungal sequences comprised less than 0.5% of the soil data set, with most attributable to vascular plants. Our results suggest that high-throughput methods can produce fairly accurate estimates of fungal abundances in complex communities. Further improvements might be achieved through corrections for rRNA copy number and utilization of standardized mock communities.IMPORTANCEFungi play numerous important roles in the environment. Improvements in sequencing methods are providing revolutionary insights into fungal biodiversity, yet accurate estimates of the number of fungal species (i.e., richness) and their relative abundances in an environmental sample (e.g., soil, roots, water, etc.) remain difficult to obtain. We present improved methods for high-throughput Illumina sequencing of the species-diagnostic fungal ribosomal marker gene that improve the accuracy of richness and abundance estimates. The improvements include new PCR primers and library preparation, validation using a known mock community, and bioinformatic parameter tuning.

scholarly journals Restructuring of the Aquatic Bacterial Community by Hydric Dynamics Associated with Superstorm Sandy

2016 ◽  
Vol 82 (12) ◽  
pp. 3525-3536 ◽  
Author(s):  
Nikea Ulrich ◽  
Abigail Rosenberger ◽  
Colin Brislawn ◽  
Justin Wright ◽  
Collin Kessler ◽  
...  
Keyword(s):  
Community Structure ◽  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Bacterial Community Structure ◽  
High Throughput Sequencing ◽  
Storm Event ◽  
Rrna Gene ◽  
Content Type

ABSTRACTBacterial community composition and longitudinal fluctuations were monitored in a riverine system during and after Superstorm Sandy to better characterize inter- and intracommunity responses associated with the disturbance associated with a 100-year storm event. High-throughput sequencing of the 16S rRNA gene was used to assess microbial community structure within water samples from Muddy Creek Run, a second-order stream in Huntingdon, PA, at 12 different time points during the storm event (29 October to 3 November 2012) and under seasonally matched baseline conditions. High-throughput sequencing of the 16S rRNA gene was used to track changes in bacterial community structure and divergence during and after Superstorm Sandy. Bacterial community dynamics were correlated to measured physicochemical parameters and fecal indicator bacteria (FIB) concentrations. Bioinformatics analyses of 2.1 million 16S rRNA gene sequences revealed a significant increase in bacterial diversity in samples taken during peak discharge of the storm. Beta-diversity analyses revealed longitudinal shifts in the bacterial community structure. Successional changes were observed, in whichBetaproteobacteriaandGammaproteobacteriadecreased in 16S rRNA gene relative abundance, while the relative abundance of members of theFirmicutesincreased. Furthermore, 16S rRNA gene sequences matching pathogenic bacteria, including strains ofLegionella,Campylobacter,Arcobacter, andHelicobacter, as well as bacteria of fecal origin (e.g.,Bacteroides), exhibited an increase in abundance after peak discharge of the storm. This study revealed a significant restructuring of in-stream bacterial community structure associated with hydric dynamics of a storm event.IMPORTANCEIn order to better understand the microbial risks associated with freshwater environments during a storm event, a more comprehensive understanding of the variations in aquatic bacterial diversity is warranted. This study investigated the bacterial communities during and after Superstorm Sandy to provide fine time point resolution of dynamic changes in bacterial composition. This study adds to the current literature by revealing the variation in bacterial community structure during the course of a storm. This study employed high-throughput DNA sequencing, which generated a deep analysis of inter- and intracommunity responses during a significant storm event. This study has highlighted the utility of applying high-throughput sequencing for water quality monitoring purposes, as this approach enabled a more comprehensive investigation of the bacterial community structure. Altogether, these data suggest a drastic restructuring of the stream bacterial community during a storm event and highlight the potential of high-throughput sequencing approaches for assessing the microbiological quality of our environment.

scholarly journals Composition and Origin of the Fermentation Microbiota of Mahewu, a Zimbabwean Fermented Cereal Beverage

2019 ◽  
Vol 85 (11) ◽  
Author(s):  
Felicitas Pswarayi ◽  
Michael G. Gänzle
Keyword(s):  
16S Rrna ◽  
Quantitative Pcr ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Starter Cultures ◽  
Rrna Gene ◽  
Culture Methods ◽  
Content Type ◽  
Cell Counts

ABSTRACTMahewu is a fermented cereal beverage produced in Zimbabwe. This study determined the composition and origin of mahewu microbiota. The microbiota of mahewu samples consisted of 3 to 7 dominant strains of lactobacilli and two strains of yeasts.Enterobacteriaceaewere not detected.Candida glabratawas present in high cell counts from samples collected in summer but not from samples collected in winter. Millet malt is the only raw ingredient used in the production of mahewu and is a likely source of fermentation microbiota; therefore, malt microbiota was also analyzed by culture-dependent and high-throughput 16S rRNA gene sequencing methodologies. Millet malt contained 8 to 19 strains ofEnterobacteriaceae, lactobacilli, bacilli, and very few yeasts. Strain-specific quantitative PCR assays were established on the basis of the genome sequences ofLactobacillus fermentumFUA3588 and FUA3589 andLactobacillus plantarumFUA3590 to obtain a direct assessment of the identity of strains from malt and mahewu.L. fermentumFUA3588 and FUA3589 were detected in millet malt, demonstrating that millet malt is a main source of mahewu microbiota. Strains which were detected in summer were not detected in samples produced at the same site in winter. Model mahewu fermentations conducted with a 5-strain inoculum consisting of lactobacilli,Klebsiella pneumoniae,andCronobacter sakazakiidemonstrated that lactobacilli outcompeteEnterobacteriaceae, which sharply decreased in the first 24 h. In conclusion, mahewu microbiota is mainly derived from millet malt microbiota, but minor components of malt microbiota rapidly outcompeteEnterobacteriaceaeandBacillusspecies during fermentation.IMPORTANCEThis study provides insight into the composition and origin of the microbiota of mahewu and the composition of millet malt microbiota. Fermentation microbiota are often hypothesized to be derived from the environment, but the evidence remains inconclusive. Our findings confirm that millet malt is the major source of mahewu microbiota. By complementing culture methods with high-throughput sequencing of 16S rRNA amplicons and strain-specific quantitative PCR, this study provides evidence about the source of mahewu microbiota, which can inform the development of starter cultures for mahewu production. The study also documents the fate ofEnterobacteriaceaeduring the fermentation of mahewu. There are concerns regarding the safety of traditionally prepared mahewu, and this requires in-depth knowledge of the fermentation process. Therefore, this study elucidated millet malt microbiota and identified cultures that are able to control the high numbers ofEnterobacteriaceaethat are initially present in mahewu fermentations.

scholarly journals Novel Library Method for High-Throughput Full-Length Sequencing of the Immune Repertoire from Unseparated B-Cells with Single-Cell Resolutionution

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3699-3699
Author(s):  
Stefano Vergani ◽  
Ilya Korsunsky ◽  
Nicholas Chiorazzi ◽  
Davide Bagnara
Keyword(s):  
B Lymphocytes ◽  
High Throughput ◽  
Genetic Material ◽  
Pcr Amplification ◽  
Full Length ◽  
Read Length ◽  
Library Preparation ◽  
Sequencing Platform ◽  
Mrna Molecule ◽  
High Throughput Dna Sequencing

Abstract High-throughput DNA sequencing of the adaptive immune receptor repertoire is a relatively new and fast growing technology used to study the immune response in health and disease. In B and T cell lymphoproliferative disorders, antigen receptor sequencing can be used to study clonal diversity and evolution of the disease in treatment free condition and in response to treatment. Furthermore, it can be used for the detection of minimal residual disease (MRD), providing information on the relationship between the presence and number of pre-treatment clone(s) and their relationship and responsibility for a subsequent relapse. The characteristics and quality of the data generated by high-throughput DNA sequencing of immune receptor signatures are the results of three major components: library preparation, sequencing platform, and software tools. For both the library and software, there are no standard protocols and tools. Indeed, new approaches are continually being developed to accommodate new sequencing platform features and shortcomings, such as errors and read length restrictions. Two major technical challenges are: procuring an unbiased repertoire library that for B lymphocytes obtains and retains the full length IGHV-D-J along with (sub)isotype information, and resolving data to a single cell level, crucial for detection of MRD and rare clonal variants existing in the early phase of the disease, which might emerge and be involved in future relapse or progression. We describe here a library preparation method for use with the Illumina MiSeq platform that results in an exhaustive full-length repertoire where virtually every B cell is sequenced, thereby maximizing the likelihood of identifying and quantifying the “real” IGHV-D-J repertoire of the sample analyzed. The method also allows the detection of very infrequent rearrangements and maintains IG sub-isotype information without compromising data quality. From 0.5 - 1 million human B cells can be sequenced in a single MiSeq 2x300 run with this approach. Key aspects of the technique are: 1) start from a well defined number of B lymphocytes 2) avoid V-gene specific PCR amplification and genetic material dilution in the pre-amplification phases 3) the specific depth of sequencing should depend on the starting B (or T) cell subset (i.e. na•ve, memory or plasma cell), and should be proportional to the number of starting cells. High quality sub-isotype information can be obtained with a second round of sequencing of shorter read length, e.g., with the Illumina 2x150 platform. We used 58 different CLL clones with known IGH sequence mixed all together with polyclonal B cell from a donor PBMC (Figure 1). The mixed lysate is used to test the ability to detect the different clones. The following describes how the absence of genetic material dilution in the pre-amplification phases impact on the ability to obtain a comprehensive repertoire. These are crucial in MRD detection, since diluting the genetic material (RNA and/or cDNA) prior PCR amplification compromises the ability to accurately and consistently detect the clonal variants, reducing the de facto sensitivity and reproducibility of the analysis. As a final example of the method's utility, we also demonstrate how different chronic lymphocytic leukemia clones present considerable variability in IG mRNA expression level that correlate with the number of unique mRNA molecule sequenced (Figure 3), which, if using a method with sub-optimal efficiency, could lead to a reduced clone-specific ability of detection by PCR based techniques. Figure 1. Figure 1. Figure 2. Each dilution is performed in replicates. The cDNA is obtained from all the RNA extracted from the starting cells. Each slice represents a different CLL, and each slice size is the frequency for which it is detected. A comprehensive detection of each CLL is dependent to the absence of genetic material dilution. Figure 2. Each dilution is performed in replicates. The cDNA is obtained from all the RNA extracted from the starting cells. Each slice represents a different CLL, and each slice size is the frequency for which it is detected. A comprehensive detection of each CLL is dependent to the absence of genetic material dilution. Figure 3. qPCR IgH expression correlate with the number of unique mRNA molecule sequenced. Figure 3. qPCR IgH expression correlate with the number of unique mRNA molecule sequenced. Disclosures No relevant conflicts of interest to declare.

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