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Methods

Evaluation of a Transposase Protocol for Rapid Generation of Shotgun High-Throughput Sequencing Libraries from Nanogram Quantities of DNA

Rachel Marine, Shawn W. Polson, Jacques Ravel, Graham Hatfull, Daniel Russell, Matthew Sullivan, Fraz Syed, Michael Dumas, K. Eric Wommack
Rachel Marine
University of Delaware, Delaware Biotechnology Institute, Newark, Delaware 19711
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Shawn W. Polson
University of Delaware, Delaware Biotechnology Institute, Newark, Delaware 19711
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Jacques Ravel
University of Maryland School of Medicine, Institute for Genome Sciences, Baltimore, Maryland 21201
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Graham Hatfull
University of Pittsburg, Department of Biological Sciences, Pittsburgh, Pennsylvania 15260
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Daniel Russell
University of Pittsburg, Department of Biological Sciences, Pittsburgh, Pennsylvania 15260
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Matthew Sullivan
University of Arizona, Ecology and Evolutionary Biology Deptartment, Tucson, Arizona 85721
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Fraz Syed
Epicenter Biotechnologies, Madison, Wisconsin 53713
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Michael Dumas
University of Delaware, Delaware Biotechnology Institute, Newark, Delaware 19711
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K. Eric Wommack
University of Delaware, Delaware Biotechnology Institute, Newark, Delaware 19711
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  • For correspondence: wommack@dbi.udel.edu
DOI: 10.1128/AEM.05610-11
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  • Fig. 1.
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    Fig. 1.

    Overview of sequencing strategies used to prepare viral genomic DNA samples for 454 sequencing.

  • Fig. 2.
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    Fig. 2.

    Linear regression analysis of the percent GC content of the largest contig versus average read length for nine unknown genomes and the mock metagenome. The percent GC content for the mock metagenome represents the collective percent GC for all nine reference genomes.

  • Fig. 3.
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    Fig. 3.

    Comparison of the predicted sequence coverage of each member of the mock metagenome to the experimental coverage (± SD). The predicted coverage was normalized to the experimental average read length and the number of sequence reads obtained after quality screening.

  • Fig. 4.
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    Fig. 4.

    Box-and-whisker plots of the sequence coverage range as a function of phage genome GC content. Phage names are provided above each panel. (a) The Roche GS FLX Titanium general library preparation method. (b) The Nextera method with high-GC-content phage. (c) The Nextera method with low-GC-content phage. Whiskers represent the 5th and 95th percentiles; open diamonds represent mean values.

  • Fig. 5.
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    Fig. 5.

    Average GC content (± SD) of regions with low or high coverage in comparisons of individual phage genomes. (a) Phage comprising the mock metagenome and unknown phage genomes prepared using the Nextera protocol. (b) Mycobacteriophage genomes prepared using the Roche GS FLX Titanium general library preparation method. Values in parentheses after the phage name indicate the average % GC content of the genome. Classification of the results into low- and high-coverage areas corresponded to the regions above or below 1.5 standard deviations from the average coverage. Genomes with at least 5 regions of low and high coverage of 50 bp or longer were included in the analysis.

Tables

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  • Table 1.

    DNA yields and fragmentation results after completion of the Nextera protocol

    PhageTypeaFinal DNA concn (ng/μl)Total DNA (ng)bConcn fragments of 300–800 bpc (ng/μl)% total DNA (300–800 bp)dFragment size of greatest abundancec (bp)Avg read length for 454 sequences (bp)e
    AngelicaM12307979569373
    AthenaM3385827811,402382
    AvrafanM213221149843355
    Blue7M13332968398336
    WeeM194821265529348
    TUSD 1Cy3491520581,392320
    TUSD 14Cy2157111511,348307
    TUSD 20Cy205361052732317
    TUSD 21Cy2977811391,359315
    Mock metagenome2270812551,151349
    • ↵a M, mycobacteriophage; Cy, cyanophage.

    • ↵b x̅ ± SD = 581 ± 226 ng.

    • ↵c Data were determined using an Agilent Bioanalyzer.

    • ↵d x̅ = 60%.

    • ↵e The average read lengths were calculated after removal of sequences of less than 100 bp.

  • Table 2.

    Results determined for de novo assemblies of 454 pyrosequencing reads from Nextera and LASL fragment libraries

    PhageNo. of readsNo. of contigsN50 contig (bp)N90 contig (bp)Major contig(s) of alignmenta
    Before cleaningAfter cleaningBefore cleaningAfter cleaningSize(s) (bp)No. of readsAvg coveragec (± SD)Maximum coveragecPairwise identity (%)GC (%)
    Angelica4,9083,96811728,45218,19628,4521,67722 (12)7399.566.9
    18,1961,39629 (15)7393.165.8
    Athena18,59915,198186569,41069,41069,41015,18084 (39)25399.167.5
    Avrafan15,56212,9754241,90241,90241,90212,970111 (60)34999.466.6
    Blue732,01025,495115152,23352,23352,23325,490165 (54)38199.461.3
    Wee18,52516,01212259,24559,24559,24516,00495 (39)27399.461.8
    TUSD 1b13,7639,94227215837,94437850,727, 37,9445,665, 3,43536 (11), 29 (11)84, 7099.4, 99.436.7, 37.3
    TUSD 1415,3457,5681,13920149735038,1786,62855 (16)10899.437.3
    TUSD 20 (Nextera)18,06614,311533138,09844338,09814,151118 (31)21099.337.3
    TUSD 20 (LASL)12,7058,855–207,6107267,6101,00526 (13)6799.038.0
    TUSD 21b (Nextera)16,06111,18488536,669, 32,801465, 31536,669, 37,6269,403, 1,75481 (27), 15 (6)173, 3599.1, 99.435.9, 37.3
    TUSD 21b (LASL)31,06920,5886237,873, 32,80112,916, 6,64071 (188), 39 (27)2,326, 22199.4, 98.937.2, 35.7
    • ↵a For genomes with two or more major contigs, the information on the second contig is also listed.

    • ↵b For TUSD phages, host DNA contamination was removed using cross_match.

    • ↵c Coverage is defined as number of bases per position in the consensus sequence.

  • Table 3.

    Predicted versus experimental coverage and percent abundance of reads for each phage in the mock metagenome

    PhageVirus typeaGenome size (bp)GC content (%)Predicted resultExperimental result
    Recruitment to reference sequenceMock assembly
    No. of readsCoverageAbundance of reads (%)No. of readsCoverageb (± SD)Abundance of reads (%)No. of contigsLongest contig (bp)N50 contig (bp)
    T4C166,0003520,0004728.131,04961.6 (14.6)48.23169,170169,170
    CateraM153,7666514,0003619.77,59218.0 (8.6)11.8485,29885,298
    FruitloopM58,4716212,0008216.88,74553.3 (18.9)13.6158,30858,308
    GumballM64,8076010,0006214.09,99956.4 (18.8)15.5164,80764,807
    OmegaM110,865618,0002911.23,36210.9 (5.2)5.2641,62620,153
    PorkyM76,312634,000215.62,0389.7 (5.8)3.2734,43417,944
    SolonM49,487642,000162.87155.2 (3.4)1.11210,5707,363
    P-SS2Cy107,530521,0003.71.47572.5 (2.2)1.2812,9241,313
    S-SM1Cy174,079412000.50.32050.4 (0.9)0.350915463
    Total71,20064,718 (257 unassigned, 64,462 assigned)172 (6 unassigned, 166 assigned)
    • ↵a C, coliphage; M, mycobacteriophage; Cy, cyanophage.

    • ↵b Coverage is defined as number of bases per position in the consensus sequence.

Additional Files

  • Figures
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  • Supplemental Material

    Files in this Data Supplement:

    • Supplemental Figure 1 - Histogram of the relative frequency of fragments sizes for mycobacteriophage Athena genomic DNA (Fig. S1), linear regression analysis of the percent GC of the largest contig versus the average read length for the nine phage that compose the mock metagenome (Fig. S2), length distribution of assembled sequencing reads generated from the Nextera system before quality screening (Fig. S3), identical sites and pairwise identities of mock metagenome reads which recruited to phage T4, Catera, Gumball, and P-SS2 (Table S1), and comparison of the listed genome lengths for Athena, Avrafan, Blue7, and Wee on the Mycobacteriophage database to the consensus sequence length of the major contig generated from de novo assembly (Table S2).
      PDF file, 1.2 MB.
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Evaluation of a Transposase Protocol for Rapid Generation of Shotgun High-Throughput Sequencing Libraries from Nanogram Quantities of DNA
Rachel Marine, Shawn W. Polson, Jacques Ravel, Graham Hatfull, Daniel Russell, Matthew Sullivan, Fraz Syed, Michael Dumas, K. Eric Wommack
Appl. Environ. Microbiol. Nov 2011, 77 (22) 8071-8079; DOI: 10.1128/AEM.05610-11

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Evaluation of a Transposase Protocol for Rapid Generation of Shotgun High-Throughput Sequencing Libraries from Nanogram Quantities of DNA
Rachel Marine, Shawn W. Polson, Jacques Ravel, Graham Hatfull, Daniel Russell, Matthew Sullivan, Fraz Syed, Michael Dumas, K. Eric Wommack
Appl. Environ. Microbiol. Nov 2011, 77 (22) 8071-8079; DOI: 10.1128/AEM.05610-11
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