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

63966271843.7642.3943.1243.04

639662717206.89203.14204.39202.81

639662716133.06130.97132.18132.17

639662715537.67521.59530.32539.23

639662714716.31725.12717.34718.54

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

63966269012.1911.9212.0911.68

639662689150.19148.02152.37152.32

639662688146.11139.18143.9144.11

63966268766.7965.566.8366.88

639662686411.7428.69420.77418.85

639662685114.89107.8111.55114.08

639662684151.78151.73152151.42

63966268384.1486.3285.5485.06

6396626821635.631652.531636.221644.42

6396626817.667.37.777.86

63966268040.4139.0140.8840.87

639662679263.46253.46259.37263.99

639662671164.15162.68161.27158.59

6396626703.543.263.413.35

63966266930.2330.3230.2729.65

63966266682.3380.5682.0682.98

639662665943.59915.38935.09955.68

639662664598.36582.41591.08602.38

63966266345.5546.8946.547.28

639662662311.65310.3311.29311.48

639662661421.77424.29420.54420.99

639662660778.76759.6778.54777.18

639662659191.8188.99189.33191.04

639662658402.77391.4396.77398.88

639662657100.6596.5898.4698.7

63966265510812.2210842.6210766.5210785.67

6396626548355.358463.368338.848313.97

63966265316932.3817124.9516869.9616885.96

639662652366.78345.06354.08359.43

639662651834.18800.85822.06838.74

639662650235.53227.31231.88236.97

639662649107.88109.13108.27109.94

63966264857.8558.2657.4857.8

63966264736.5836.9336.6636.16

63966264620.2820.4620.6220.81

63966264535.8235.335.4235.64

6396626442354.922341.842342.52341.95

63966264386.4785.9987.3187.31

63966264222.5721.2422.922.23

63966264115.0314.9915.9415.78

63966264093.1390.8493.4895.32

639662639165.88163.92168.64166.53

639662638698.02708.04717.94702.99

639662637107.63107.14109.14108.3

63966263637.5837.3537.738.12

639662635155.21153.96154.74156.97

6396626340.960.9711.01

63966263361.1261.6460.861.39

63966263250.5451.2950.1950.2

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

639662626166.19167.57165.19164.34

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

639662616408.58413.88424.76414.57

639662615151.84151.97159.8153.07

639662614535.12533.93544.11538.94

639662613617.2598.86610614.46

639662612928.16928.77928.39932.94

63966261163.5565.0266.4265

63966261084.1783.7885.8886.04

639662609221.94214.78221.3221.1

639662608459.51454.04463.29463.65

639662607513.68494.19505.14512.44

63966260658.5158.4158.3257.86

63966260560.5560.4662.4763.76

6396626043.323.293.353.47

6396626037.947.977.837.63

6396626024.034.374.254.48

6396626010.770.720.770.81

6396626001.531.571.611.53

6396625995.454.955.425.35

6396625987.126.857.167.31

6396625972.272.112.212.28

6396625962.52.22.282.2

63966259514.2913.5814.0614.16

63966259410.8910.2610.4510.17

6396625939.9310.029.989.83

6396625921261.811273.531267.841247.02

63966259129.8128.4828.8628.84

63966259047.8748.8849.1549.57

639662589329.12327.44326.76330.58

6396625882894.352943.052915.542833.18

639662587201.3195.78201.25204.08

639662586128.73118.36126.42127.95

639662585454.75460.01470.76458.56

639662584152.6146.66152.23154.02

639662583146.17137.58142.86145.81

6396625821936.351959.731931.511951.02

63966258119.4518.9519.3219.54

63966258022.9421.9322.6322.92

63966257991.1286.9889.2990.52

6396625781505.711464.421486.631505.55

639662577709.02693.8705.15705.36

63966257656.3855.8555.9156.68

639662575155.24151.7154.31156.62

639662574142.22144.49142.49143.56

639662573157.51153.15155.48157.55

6396625720000

63966256587.8381.6487.8589.65

6396625641640.531534.111610.041636.85

6396625632028.442026.112066.062033.45

6396625623450.543485.13445.733443.71

639662561263.35254.67260.51264.43

639662560414.73415.09418.5414.05

639662559600.86592.91592.69585.76

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6378003902.132.412.212.08

6378003890000

6378003870.280.240.270.26

6378003861.871.871.811.76

6378003851.51.631.611.73