Open Access Highly Accessed Research article

RNA-seq: technical variability and sampling

Lauren M McIntyre1*, Kenneth K Lopiano2, Alison M Morse1, Victor Amin1, Ann L Oberg3, Linda J Young2 and Sergey V Nuzhdin4

Author Affiliations

1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, USA

2 Department of Statistics, University of Florida, Gainesville, Florida, USA

3 Department of Health Sciences Research, Division of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, Minnesota, USA

4 Molecular and Computational Biology, University of Southern California, California, USA

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BMC Genomics 2011, 12:293  doi:10.1186/1471-2164-12-293

Published: 6 June 2011

Additional files

Additional file 1:

Overlapping exons combined into single genomic region. D. melanogaster ovo gene (Flybase ID BFgn0003028) used as an example of combining overlapping exons into a single genome region for mapping purposes. Format PDF. View with Adobe.

Format: PDF Size: 93KB Download file

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Additional file 2:

Chromosome postions for overlapping exons combined into single genomic regions. Format TSV. View with Wordpad.

Format: TSV Size: 2.9MB Download file

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Additional file 3:

Perl script for mapping between exons and a genome region. Format PL. View with Wordpad.

Format: PL Size: 2KB Download file

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Additonal File 4:

Perl script that converts contiguous sequences in a SAMtools consensus pileup to FASTA, BED, or coordinate tables. Format PL. View with Wordpad.

Format: PL Size: 5KB Download file

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Additional file 5:

Perl script to find common sequences from contig BEDs. Format PL. View with Wordpad.

Format: PL Size: 3KB Download file

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Additional file 6:

St_2a_D.melanogaster_common_contigs.bed. BED file containing chromosome postion, start, end and common_contig_ID for D. melanogaster experiment (Experiment 1). Format BED. View with Wordpad.

Format: BED Size: 704KB Download file

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Additional file 7:

St_2b_Dsimulans_common_contigs.bed. BED file containing chromosome postion, start, end and common_contig_ID for D. simulans (Experiment 2). Format BED. View with Wordpad.

Format: BED Size: 800KB Download file

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Additional file 8:

St_2c_167_common_contigs.bed. BED file containing chromosome postion, start, end and common_contig_ID for c167 experiment (Experiment 3). Format BED. View with Wordpad.

Format: BED Size: 759KB Download file

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Additional file 9:

Agreement between technical replicates and biological replicates for RPKM measured on common contigs. A comparison in the agreement of the estimation of the amount of expression. Agreement in whether common contigs are expressed, expressed at a low level (0 < RPKM < 20), and expressed at a high level (RPKM > 20) and agreement on a 9 level ordinal scale as follows: RPKM less than 10, 20, 40, 80, 160, 320, 1000 and greater than 1000. Agreement was measured using a kappa coefficient. The number of common contigs where disagreement is greater than 2 logs and greater than one log are also given. Format XLS. View with Excel.

Format: XLSX Size: 10KB Download file

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Additional file 10:

Length of the Exon does not explain disagreement in technical replicates. Coverage plots of the c167 cell lines data. The Y axis is the average coverage across all technical replicates. A bar is drawn if the exon is present (at any coverage level) in that technical replicate. The 10th percentile represents that bottom 10% of the exons in length while the 90th percentile represents the top 10% of exons by length. Format PDF. View with Adobe

Format: PDF Size: 745KB Download file

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Additional file 11:

Bland Altman plots for each biological replicate.

Format: PDF Size: 4.5MB Download file

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