ERCC Data Analysis - exRNA Portal€¦ · ERCC Data Analysis 1 Williams Z., et al. (2013) Comprehensive proﬁling of circulating microRNA via small RNA sequencing of cDNA libraries

Use Case 3: Biomarker Potential and Limitations of Circulating miRNA

Performed by the Data Management and Resource Repository

(DMRR)

ERCC Data Analysis

1Williams Z., et al. (2013) Comprehensive profiling of circulating microRNA via small RNA sequencing of cDNA libraries reveals biomarker potential and limitations. PNAS 110: 4255–4260.


Williams Z., et al. (2013) Comprehensive profiling of circulating microRNA via small RNA sequencing of cDNA libraries reveals biomarker potential and limitations. PNAS 110: 4255–4260. 2

The goal of this use case is to show that the Genboree Workbench and the exceRpt small RNA-seq analysis pipeline can replicate the results of Williams et al. We have developed these pipelines because as the Extracellular RNA Communication Consortium (ERCC) begins to generate more datasets, it is vital that they be analyzed in a reproducible and comparable way.

The dataset from Williams et al is freely available in the Short Read Archive (SRA), so it makes a good practice dataset for becoming familiar with the Workbench and exceRpt.

Biological Question of Interest

3

This work from the Tuschl lab at Rockefeller University is mainly a technical study. They examined extracellular RNA from the bloodstream of mothers and fathers, their newborn babies, and the placenta. They were interested in quantitating how much exRNA could be found, and whether it would be possible to detect biomarker RNAs at that level. The placenta acted as a model of a tumor; their thinking was that if detecting exRNAs from placenta is feasible, the same should be true of tumors.


Williams Z., et al. (2013) Comprehensive profiling of circulating microRNA via small RNA sequencing of cDNA libraries reveals biomarker potential and limitations. PNAS 110: 4255–4260.

Biological Samples to Be Analyzed


4

UniqueID

SampleName SRAID SampleDescription

31 C01 SRR822433 Non-pregnantwomanplasmaSample#132 C02 SRR822461 Non-pregnantwomanplasmaSample#233 C03 SRR822434 Non-pregnantwomanserumSample#334 C04 SRR822435 Non-pregnantwomanserumSample#435 C05 SRR822436 Non-pregnantwomanserumSample#518 F01 SRR822466 Father'sbloodfromSet#119 F06 SRR822441 Father'sbloodfromSet#620 F08 SRR822442 Father'sbloodfromSet#821 F10 SRR822443 Father'sbloodfromSet#1022 F12 SRR822440 Father'sbloodfromSet#121 M01 SRR822467 Mother'sbloodfromSet#12 M02 SRR822468 Mother'sbloodfromSet#23 M03 SRR822469 Mother'sbloodfromSet#34 M06 SRR822445 Mother'sbloodfromSet#65 M08 SRR822446 Mother'sbloodfromSet#86 M10 SRR822447 Mother'sbloodfromSet#107 M12 SRR822448 Mother'sbloodfromSet#1223 P05 SRR822452 PlacentafromSet#524 P06 SRR822453 PlacentafromSet#625 P07 SRR822454 PlacentafromSet#726 P08 SRR822455 PlacentafromSet#827 P09 SRR822456 PlacentafromSet#928 P10 SRR822457 PlacentafromSet#1029 P11 SRR822458 PlacentafromSet#1130 P12 SRR822459 PlacentafromSet#128 B01 SRR822462 UmbilicalcordbloodfromSet#19 B02 SRR822464 UmbilicalcordbloodfromSet#210 B03 SRR822464 UmbilicalcordbloodfromSet#311 B04 SRR822465 UmbilicalcordbloodfromSet#412 B06 SRR822437 UmbilicalcordbloodfromSet#613 B07 SRR822450 UmbilicalcordbloodfromSet#714 B08 SRR822438 UmbilicalcordbloodfromSet#815 B10 SRR822439 UmbilicalcordbloodfromSet#1016 B11 SRR822451 UmbilicalcordbloodfromSet#1117 B12 SRR822444 UmbilicalcordbloodfromSet#12

UniqueID



UniqueID



The dataset analyzed in this use case is available from the Short Read Archive (SRA) under Bioproject PRJNA187509.

http://www.ncbi.nlm.nih.gov/bioproject/PRJNA187509

To match the sample names from the article with SRA accessions from the exceRpt output, we visit the Bioproject page at the SRA:http://www.ncbi.nlm.nih.gov/bioproject/PRJNA187509

Use Case 3: Data Scrubbing


To match the sample names from the article with SRA accessions from the exceRpt output, we visit the Bioproject page at the SRA:http://www.ncbi.nlm.nih.gov/bioproject/PRJNA187509





After matching sample IDs between the two pipelines, we can import both tables into Excel and start generating comparison plots.


Use Case 3: Data Comparison

Results from the Article’s analysis pipeline



We can compare the number of reads mapped to various categories of RNA in Table S1 from the article with the same data from the exceRpt read mapping summary. Categories in common are total input reads, microRNA, ribosomal RNA, and transfer RNA.



0

2,000,000

4,000,000

6,000,000

8,000,000

10,000,000

12,000,000

14,000,000

C01

C02

C03

C04

C05 F01

F06

F08

F10

F12

M01

M02

M03

M06

M08

M10

M12 P05

P06

P07

P08

P09

P10

P11

P12

B01

B02

B03

B04

B06

B07

B08

B10

B11

B12

Num

ber o

f Rea

ds

Sample

Article exceRpt 11

Use Case 3: exceRpt Pipeline Results -Number of Input Reads

0

2,000,000

4,000,000

6,000,000

8,000,000

10,000,000

12,000,000

C01

C02

C03

C04

C05 F01

F06

F08

F10

F12

M01

M02

M03

M06

M08

M10

M12 P05

P06

P07

P08

P09

P10

P11

P12

B01

B02

B03

B04

B06

B07

B08

B10

B11

B12

Num

ber o

f Rea

ds

Sample

Article exceRpt 12

Use Case 3: exceRpt Pipeline Results – MicroRNA

-

200,000

400,000

600,000

800,000

1,000,000

1,200,000

1,400,000

1,600,000

1,800,000

2,000,000

C01

C02

C03

C04

C05 F01

F06

F08

F10

F12

M01

M02

M03

M06

M08

M10

M12 P0

5P0

6P0

7P0

8P0

9P1

0P1

1P1

2B0

1B0

2B0

3B0

4B0

6B0

7B0

8B1

0B1

1B1

2

Num

ber o

f Rea

ds

Sample

Article exceRpt 13

Use Case 3: exceRpt Pipeline Results -Ribosomal RNA

14

Use Case 3: exceRpt Pipeline Results -Transfer RNA

-

50,000

100,000

150,000

200,000

250,000

300,000

350,000

C01

C02

C03

C04

C05 F01

F06

F08

F10

F12

M01

M02

M03

M06

M08

M10

M12 P0

5P0

6P0

7P0

8P0

9P1

0P1

1P1

2B0

1B0

2B0

3B0

4B0

6B0

7B0

8B1

0B1

1B1

2

Num

ber o

f Rea

ds

Sample

Article exceRpt

The exceRpt analysis pipeline recapitulates the analysis from Williams et al. except for significant differences in the number of reads mapped to transfer RNA.

Use Case 3: Summary

15http://genboree.org/theCommons/projects/exrna-tools-may2014/wiki/Version_Updates

We used exceRpt version 2.8.8; the latest version is 3.1.9. At the Genboree Workbench there is a version history.

Use Case 3: Summary

16http://genboree.org/theCommons/projects/exrna-tools-may2014/wiki/Version_Updates

v2.2.8

•  We moved the alignment against endogenous repetitive elements (RE) to occur after the main smallRNA alignments performed by sRNABench. This is because we noticed that the RE library was able to ‘compete’ for reads that would be better annotated/interpreted as coming from tRNAs, piRNAs, or other transcripts. This competitive alignment did not ever affect miRNAs as these are always aligned to before other annotated RNAs, but we expect that this update will faithfully capture reads aligning to repetitive small-RNAs, especially tRNAs, piRNAs, and snoRNAs.

•  exceRpt still aligns to REs as a final step before aligning to exogenous sequences as this is critical to remove highly repetitive endogenous sequences that might otherwise be confused as exogenous sequences.

Acknowledgements

17

BaylorCollegeofMedicine,Houston,TXAleksandarMilosavljevicMa;hewRothGenboreeTeamatBaylorAndrewJacksonSaiLakshmiSubramanianWilliamThistlethwaiteSameerPaithankarNeethuShahAaronBakerYaleUniversity,NewHaven,CTMarkGersteinJoelRozowskyRobKitchenFabioNavarroGladstoneInsAtuteAlexanderPicoAndersRiu;a

PacificNorthwestDiabetesResearchInsAtute,SeaHle,WADavidGalasRogerAlexanderRockefellerUniversity,NewYork,NYTomTuschlNIHJohnSa;erleeLillianKuoDenaProcaccini

Use Case 3: References

18

1.  Williams Z., et al. (2013) Comprehensive profiling of circulating microRNA via small RNA sequencing of cDNA libraries reveals biomarker potential and limitations. PNAS 110: 4255–4260.

2.  Farazi T.A., et al. (2012) Bioinformatic analysis of barcoded cDNA libraries for small RNA profiling by next-generation sequencing. Methods 58: 171-187.

3.  ExRNA Portal Software Resources (http://exrna.org/resources/software)

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Useful Links

•  exRNA Portal Software Resources – http://exrna.org/resources/software

•  exRNA Atlas – http://genboree.org/exRNA-atlas/

•  Genboree Workbench – http://genboree.org/java-bin/workbench.jsp

•  Data Coordination Center Wiki – http://genboree.org/theCommons/projects/exrna-mads/wiki

•  exRNA Data Analysis Tools Wiki – http://genboree.org/theCommons/projects/exrna-tools-may2014/wiki

•  Use Case Tutorials – exRNA Portal Data Resource http://exrna.org/resources/data/

Documents

ERCC Data Analysis - exRNA Portal€¦ · ERCC Data Analysis 1 Williams Z., et al. (2013) Comprehensive proﬁling of circulating microRNA via small RNA sequencing of cDNA libraries