metatranscriptomics: challenges and progress

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Metatranscriptomics: Challenges and Progress Cindi Hoover DOE Joint Genome Institute May 17, 2012

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Metatranscriptomics: Challenges and Progress. Cindi Hoover DOE Joint Genome Institute May 17, 2012. Metatranscriptomics. Metatranscriptome The complete collection of transcribed sequences in a microbial community: Protein-coding RNA (mRNA) Non-coding RNA (rRNA, tRNA, regulatory RNA, etc) - PowerPoint PPT Presentation

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  • Metatranscriptomics:Challenges and ProgressCindi HooverDOE Joint Genome InstituteMay 17, 2012

  • MetatranscriptomicsMetatranscriptomeThe complete collection of transcribed sequences in a microbial community:

    Protein-coding RNA (mRNA) Non-coding RNA (rRNA, tRNA, regulatory RNA, etc)

    Metagenome = whos there?Metatranscriptome = function?What genes are active in environment?How does gene expression change in response to particular conditions?

  • Evolution of Metatranscriptome MethodscDNA clone libraries + Sanger sequencing (low throughput)

    Microarrays (medium throughput)

    RNA-seq enabled by next-generation sequencing technologies (high throughput)Influenced by presence of rRNA

  • Wet labLow RNA yield from environmental samplesInstability of RNA High rRNA content in total RNAmRNA = 1-5% of totalhttp://cybernetnews.com/vista-recovery-disc/http://www.nwfsc.noaa.gov/index.cfm

    BioinformaticsGeneral challenges with short reads and large data sizeSmall overlap between metagenome and metatranscriptome, or complete lack of metagenome reference

    Main ChallengesHow do you effectively removal rRNA from metatranscriptome samples?

  • rRNA Removal Methods

    MethodrRNA feature usedInput RNAManipulate raw RNABefore cDNA synthesisSubtractive hybridizationConserved sequenceHighYesRNase H digestionExonuclease digestion5 monophosphateGel extractionSizeBiased poly(A) tailing 2o structureLowDuring cDNA synthesisNot-so-random primersSequence featureLowNoAfter cDNA synthesisLibrary normalization w/ DSNHigh abundanceLowNo

  • Sample-specific probe method Stewart et al, ISME J (2010) 4, 896907One of the first to successfully tackle the rRNA in metatranscriptome problem

    PRO: Customized probes are specific to communities of interest

    CONS: Very time consuming process; requires >3ug RNA or matched DNA samples

    Different batches of probe may give different resultsMethod has been applied on marine metatranscriptome samples to substantially reduce rRNA.

  • Epicentre: Ribo-Zero TMEssentially a subtractive hybridization

    rRNA removal reagent contains oligo probes complementary to rRNA sequences

    Magnetic beads bind rRNA-probe complexes and remove them from solution

    Process takes ~1-1.5 hours; requires 1ug total RNA

  • Ribo-Zero TypesMetabacteria: handles Gram (-) and Gram (+) Human/Mouse/Rat: also works on fungal samplesPlant LeafPlant Seed/Root

  • Synthetic metatranscriptomeBoth methods tested on sample Mettr_1:

    OrganismAmount in pool (ug)Prochlorococcus marinus pastoris CMP19860.1Pediococcus pentosaceus6.0Acinetobacter sp. ADP12.5Cyanobacterium synechocystis PCC 68033.0Synechococcus elongates PCC 79420.5Total Pool12 ug

  • Example of Depletion QCRed = total RNABlue = (+) Ribo-Zero AGreen = (+) Ribo-Zero BAgilent Nano chip: total RNA vs depletion with beta test kit

  • Initial Mettr_1 Data

    SampleTotal reads (million)% rRNA% MapMettr_1 CONTROL (no depletion)6.0875.44.1Mettr_1 (+) probe6.7619.324.3Mettr_1 Ribo-zero A7.964.168.0Mettr_1 Ribo-zero B6.824.369.5

  • Gene Expression Correlations Ribo-Zero vs. No DepletionRibo-Zero does not appear cause bias in gene expression.

  • Gene Expression Ribo-Zero vs Probe Method

    All

    GENECNSY.cpmCNCB.cpmCNSX.cpmCNSZ.cpmCorrelations Across All Genes

    63966298339.0838.8938.939.86CNSX- controlCNSY-RiboZero BCNSZ-RiboZero B5CNCB-probe (+)

    63966298214.041414.314.88CNSX- control1.000000.999970.999970.99988

    63966298150.5951.3851.2250.72CNSY-RiboZero B0.999971.000000.999980.99980

    63966298085.8284.9985.4485.32CNSZ-RiboZero B50.999970.999981.000000.99979

    639662979235.05235.45236.43237.4CNCB-probe (+)0.999880.999800.999791.00000

    63966297843.3141.8242.2842.34

    639662977210.77210.23208.85209.66

    63966297638.3444.4741.1139.65

    63966297588.5786.9587.7889.02

    6396629749.38.428.978.64

    639662973395.62401.45399.78398.68

    63966297277.9979.1479.1879.02

    6396629712.842.722.682.43

    63966297035.1733.9134.6834.94

    639662969397.69391.77406.71398.82

    639662968481.86459.44484.25482.35

    63966296726.0624.7725.6125.72

    639662966766.71758.88778.2760.62

    639662965248.06247.04248.75247.09

    63966296461.0656.6960.9762.14

    639662963242.96248.7244.5243.04

    639662962108.96103.67107.13109.19

    63966296132.1932.6432.3731.62

    63966296016.0515.8415.8715.9

    6396629591.131.121.211.36

    639662958209.21211.5208.24208.56

    639662957375.97379.43378.39382.52

    6396629567.066.827.137.4

    6396629552.642.812.882.95

    6396629544.174.044.294.36

    6396629534.454.254.254.13

    63966295263.4465.1163.8163.76

    6396629512.31.992.212.28

    63966295029.1828.9929.4329.71

    63966294940.7242.3341.4141.27

    63966294844.544.843.7242.66

    6396629470.450.420.470.49

    639662946364.34369.83365.97367.09

    63966294516.7916.7416.6116.33

    6396629441.671.871.771.79

    639662943415.16413.79417.26421.19

    639662942122.2120.17124.11123.67

    6396629411217.971174.871204.161206.24

    639662940981.9950.86967.53985.16

    63966293914.381414.2613.7

    63966293811.2611.411.3511.68

    63966293727.8226.9127.6528.15

    63966293613.9813.0313.5913.82

    639662934302.15304.87302.12304.83

    639662933246.84239.43245.1247.78

    639662932151.78148.47152.9152.84

    639662931129.01123.31128.09129.45

    63966293063.4760.9763.5163.87

    639662929264.94258.2264.22263.7

    63966292889.0887.5989.4288.82

    639662927161.99160.75163.85162.72

    639662926183.83188.23187.29188.5

    639662925557.87538.88546.22552.06

    6396629244.033.774.024.1

    6396629236.896.286.866.97

    639662922178.3172.39175.87178.67

    63966292157.457.2657.3258.24

    63966292014.8614.9615.315.58

    63966291929.6630.1130.0330.46

    63966291811.0310.9210.9810.93

    639662917440.03441.31439.82437.23

    639662916104.65105.36105.13105.38

    63966291536.8736.7837.1336.88

    63966291452.5552.852.8352.86

    63966291314.6614.7214.814.88

    63966291218.4618.6218.5118.41

    63966291110.8110.9210.8810.98

    63966291030.8331.2931.6431.36

    63966290955.154.6155.7455

    639662908111.48110.64112.49114.51

    639662907132.16132.21131.91131.53

    6396629060.40.30.330.29

    63966290517.7217.7117.8417.8

    6396629048.658.098.48.64

    639662903206.06201.78203.82206.91

    639662902104.93106.53107.47108.9

    6396629013.83.863.783.7

    639662900328.63336.82333.62333.85

    639662899132.36130.04134.59131.94

    6396628986.36.226.296.39

    63966289720.7920.2120.7920.98

    6396628965.815.585.665.52

    6396628951211.9212.2212.37

    63966289413.3612.8812.9212.4

    63966289346.1445.546.2747.14

    639662892110.09107.32109.38109.71

    639662891129.09126.6128.16127.34

    63966289028.9630.2929.2929.16

    63966288914.9514.0314.514.51

    6396628880.770.840.80.78

    6396628871.051.121.071.01

    6396628864397.764412.424388.454461.47

    63966288511.1510.4411.0811.27

    6396628842364.3623332354.292352.7

    63966288339.2236.238.1738.61

    639662882338.56324333.09339.26

    63966288191.2990.4891.691.07

    63966288021.8720.3321.1921.24

    6396628797.497.367.437.34

    63966287843.9942.1843.2643.41

    639662877168.4167.54167.73168.76

    639662876876.97868.4870.81867.36

    639662875107.51107.29107.27106.16

    63966287460.1258.7459.2658.67

    6396628733.012.933.013.06

    6396628724.064.134.183.96

    63966287122.7721.9623.2723.09

    6396628709.539.029.719.6

    63966286988.7990.7290.0690.7

    63966286813.6112.2512.7612.43

    639662867169.93178.61172.29170.93

    6396628663.263.143.253.24

    63966286547.7947.0449.7848.35

    6396628641230.761260.441233.731233.78

    639662863348353.24347.95345.9

    6396628624.684.564.554.51

    63966286150.5152.0452.1651.79

    63966286075.5873.9275.6375.52

    63966285921.3821.2121.4921.91

    639662858283.43279.65283.41284.14

    639662857202.6192.97197.2199.17

    6396628561007.34993.34994.351001.49

    63966285510.559.719.849.39

    639662854219.31214.24218.42217.63

    639662853455.2441.4445.85445.38

    639662852100.8598.9399.87100.61

    639662851194.09194.12193.41190.58

    63966285059.1959.1658.9958.67

    639662849443.38461.01448.66444.81

    639662848397.43387.87393.89396.34

    639662847966.99992.34967.26961.52

    639662846313.32286.77302.12305.84

    6396628451263.461195.571230.611254.45

    6396628442.412.232.342.14

    639662843474.89472.98472.57476.02

    639662842105.75105.84105.7104.28

    6396628417.697.427.838.12

    63966284011.1210.9511.2811.73

    63966283959.158.3858.2957.46

    63966283834.5736.0834.7934.34

    63966283786.3886.5685.9486.56

    63966283665.7166.0766.1967.66

    63966283538.435.8437.0337.37

    639662834101.78102.22101.85103.27

    63966283316.5916.7716.4416.07

    6396628322.012.082.011.99

    6396628315.55.555.525.38

    6396628302.752.262.412.14

    6396628294.173.954.324.45

    6396628282.782.442.612.57

    63966282714.3813.9414.4613.82

    6396628261.931.811.871.91

    639662825111.94110.18110.18109.19

    6396628244.424.284.254.16

    63966282323.8223.4423.9723.93

    63966282213.1313.2113.4313.53

    63966282131.1730.2631.3732.83

    63966282053.653.7353.853.06

    63966281938.1236.9337.638.53

    63966281814.8614.8415.215.49

    63966281729.6129.7829.5628.58

    63966281632.2531.2632.1732.57

    63966281529.7829.6329.9329.05

    63966281466.6263.3665.0865.41

    63966281357.455.3656.6157.6

    63966281241.0140.740.8141.47

    639662811355.01362.05357.26361.37

    6396628104.063.833.853.58

    639662809909.36863.45902.01899.44

    6396628082278.972153.852285.422252.1

    639662807781.48725.3780.01769.93

    639662806249.06238.35252.8246.83

    6396628051949.511846.411939.341928.91

    6396628041414.081365.791422.551413.06

    63966280378.5378.9680.3879.94

    639662802311.16310.33315.88317.29

    63966280144.3543.2644.0643.84

    639662800122.34123.16122.27121.16

    639662799252.91237.8257.46252.63

    639662798518.05472.41509.06518.48

    639662797433.28407.94430.08435.56

    6396627962.522.662.612.69

    63966279529.4928.7228.9928.84

    639662794244.52246.43244.6246.1

    6396627931339.041357.951350.91357.25

    639662792135.3133.63134.55134.25

    63966279173.9671.1172.0571.04

    639662790113.47112.75112.26111.36

    639662789128.56128.5127.09127.03

    639662788204.84194.51201.31204.74

    639662787582.6571.01575.95582.55

    63966278635.8836.7536.3936.16

    639662785101.87101.07102.51104.11

    639662784104.798.6100.81101.16

    63966278314.6314.7214.6614.71

    639662782174.67168.59174.73176.13

    639662781149.23147.44148.11148.93

    6396627804270.174450.644311.344311.07

    63966277975.3577.5477.5776.01

    63966277873.5972.4474.1674.63

    639662777100.9695.0799.54102.66

    6396627761008.11989.96998.231002.68

    639662775535.55527.56531.82536.05

    639662774860.69817.65856.14851.06

    6396627731546.891442.881534.171545.15

    6396627721954.841762.351893.171924.37

    63966277166.1964.9666.8665.67

    6396627701321.371299.991311.61320.81

    63966276914.2615.0914.5613.93

    6396627682050.331999.252056.192059.9

    6396627672991.962871.032973.863000.81

    6396627663354.693325.43358.983389.23

    639662765481.46461.34486.23479.26

    639662764788.94743.92794.17789.9

    63966276341.2439.5241.0541.33

    639662762227.19221.12223.88224.74

    639662761491.53490.09495.83493.42

    639662760275.23278.32285.65276.13

    63966275967.9866.8667.4668.5

    639662758240.29236.78237.54240.73

    639662757998.69987.97987.951002.33

    63966275616108.5316045.3216187.2116191.9

    639662755193.02191.04192.78192.78

    639662754793.99794.9799.29802.18

    639662753604.43606.91601.6605.59

    63966275229.0728.3628.7328.24

    63966275184.4389.9489.3986.04

    63966275068.2166.569.168.7

    639662749427.72420.22429.08432.73

    63966274834.2633.4633.6133.87

    639662747131.45128.23128.86129.02

    639662746146.22143.19145.37147.92

    6396627452428.962471.362492.262440.6

    63966274419.1419.7319.3519.42

    639662743160.43155.5156.99158.01

    63966274235.0533.9735.0234.57

    63966274137.6138.3539.3737.72

    63966274063.5866.6264.3563.85

    6396627397.637.247.77.89

    6396627386.076.186.265.98

    639662737211.03210.17212.9212.4

    639662736251.15245.89249.36254.6

    63966273524.5924.5624.5424.6

    63966273475.8976.1277.3777.8

    63966273395.6697.7597.1296.76

    639662732230.42230.32230.94228.1

    63966273190.8683.0688.2988.96

    63966273085.7986.3286.4887.72

    639662729157.06152.09153.97155.23

    63966272837.0134.5236.8636.73

    6396627271592.121524.731572.371572.26

    6396627261398.031375.271396.971391.36

    639662725711.61678.9699.26714.52

    6396627242937.232862.132902.592943.9

    63966272398.9897.9397.8398.59

    639662722683.95678.72677.76681.37

    639662721606.96601.21609.4609.46

    639662720292.39292.02292.24293.3

    63966271990.3590.3689.7990.06

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    639662716133.06130.97132.18132.17

    639662715537.67521.59530.32539.23

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    63966271319.5118.3718.8218.38

    639662712207.57207.45208.65207.89

    639662711178.36178.67181.36181.25

    639662710330.02334.56329.64329.89

    6396627092687.832815.612712.692684.21

    63966270836.8439.3438.3337.69

    639662707115.45108.52112.46112.69

    63966270651.2249.9950.9651.94

    639662705432.54429.84427.64429.89

    63966270475.1573.3173.8674.16

    63966270330.8829.6930.530.58

    639662702390.51389.32391.44390.73

    639662701582.71548.17572.84572.53

    639662700265.9260.82264.56266.02

    639662699296.87289.79294.65296.19

    63966269844.4742.2743.7645.03

    639662697110.01108.01109.21107.92

    639662696413.17418.04416.75417.29

    639662695129.15132.33130.44130.26

    639662694199.71211.71204.29203.85

    639662693377.5364.67372.43379.92

    639662692292.16285.14290.74293.91

    6396626916875.227235.636973.086939.67

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    639662689150.19148.02152.37152.32

    639662688146.11139.18143.9144.11

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    639662685114.89107.8111.55114.08

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    6396626821635.631652.531636.221644.42

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    63966265510812.2210842.6210766.5210785.67

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    63966263149.5250.4550.3949.39

    639662630296.05292.62296.13294.4

    639662629403.96414.91406.21401.11

    6396626281156.091199.611171.851161.35

    639662627782.39806.73791.22785.82

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    639662625335.67321.56331.52335.93

    63966262451.1950.7851.7652.95

    63966262321.3521.0621.1921.16

    63966262235.3434.2434.8235.32

    639662621240.38249.27250.83243.88

    63966262092.293.8995.0594.57

    639662619274.24286.41282.17280.73

    63966261876.1773.1976.6376.76

    639662617132.89132.51134.92134.92

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    639662615151.84151.97159.8153.07

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    639662612928.16928.77928.39932.94

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    63966261084.1783.7885.8886.04

    639662609221.94214.78221.3221.1

    639662608459.51454.04463.29463.65

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    63966260658.5158.4158.3257.86

    63966260560.5560.4662.4763.76

    6396626043.323.293.353.47

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    6396626024.034.374.254.48

    6396626010.770.720.770.81

    6396626001.531.571.611.53

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    6396625987.126.857.167.31

    6396625972.272.112.212.28

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