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Chromosome Research

, Volume 24, Issue 4, pp 481–494 | Cite as

Data analysis algorithm for DamID-seq profiling of chromatin proteins in Drosophila melanogaster

  • Daniil A. Maksimov
  • Petr P. Laktionov
  • Stepan N. BelyakinEmail author
Original Article

Abstract

Analysis of gene expression regulation typically requires identification of genomic sites bound by regulatory proteins. For this purpose, chromatin immunoprecipitation (ChIP) and Dam identification (DamID) methods can be applied to cell lines, whole organisms, or enriched cell populations. In this work, we present modifications to the experimental DamID protocol, as well as a custom data processing algorithm, that allow to confidently identify genomic sites enriched with the proteins of interest. This algorithm is implemented in Perl and is also available as executable files, thereby making DamID analysis relatively straightforward. Finally, we demonstrate how this pipeline performs when fed with real experimental data.

Keywords

DamID-seq transcription factors chromatin profiling high-throughput sequencing peak calling 

Abbreviations

ChIP

Chromatin immunoprecipitation

DamID

Dam identification

FDR

False-positive discovery rate

FRT

Flippase recognition target

MNase

Micrococcal nuclease

MPE

Methidiumpropyl-EDTA

NGS

Next-generation sequencing

ORF

Open reading frame

qPCR

Quantitative polymerase chain reaction

UAS

Upstream activating sequence

Notes

Acknowledgments

This work was supported by the Russian Foundation for Basic Research grant 16-04-01463 in a part concerning DSX profiles analysis and grant 13-04-40087 in a part concerning COMR DamID profiling in Drosophila testis. High-throughput sequencing was performed with support from the Federal Agency for Scientific Organizations of the Russian Federation (project 0310-2014-0002). The algorithm was developed for the project supported by Russian Scientific Foundation grant 14-14-00641. Perl scripts, compiled applications for Windows, and user manual are available from https://github.com/Vift/DamID-Seq.

Supplementary material

10577_2016_9538_MOESM1_ESM.pl (4 kb)
Supplementary material 1 Perl script for “GATC_mapper” (PL 3 kb)
10577_2016_9538_MOESM2_ESM.pl (17 kb)
Supplementary material 2 Perl script for “dynSD” (PL 17 kb)
10577_2016_9538_MOESM3_ESM.pl (13 kb)
Supplementary material 3 Perl script for “Fisher_auto-FDR” (PL 13 kb)
10577_2016_9538_MOESM4_ESM.txt (9.2 mb)
Supplementary material 4 List and coordinates of all GATC-fragments in the D. melanogaster genome, Release dm6. This list is used by default. (TXT 9401 kb)
10577_2016_9538_MOESM5_ESM.txt (8.2 mb)
Supplementary material 5 List and coordinates of all GATC-fragments in the D. melanogaster genome, Release dm3. This may be used for previous genome version (see DamID-seq manual). (TXT 8424 kb)
10577_2016_9538_MOESM6_ESM.pdf (114 kb)
Supplementary material 6 DamID-seq manual with step-by-step instruction for our pipeline. (PDF 114 kb)
10577_2016_9538_MOESM7_ESM.xlsx (14 kb)
Supplementary Table 1 Normalized read counts in Dam and Dam-DSX samples in males and females around Yp1 gene. The major peak is marked with bright yellow. (XLSX 14 kb)
10577_2016_9538_Fig7_ESM.gif (245 kb)
Supplementary Figure 1

Reproducibility-based filtering algorithm for biological replicas (“dynSD” package). Each GATC-fragment is represented by a dot, whose coordinates correspond to the read numbers bound in the first and second biological replicas. Local filtering is performed in groups of 200 fragments. GATC-fragments whose reads differ by more than 2 SD from the group average, are removed from downstream analyses (red) (Materials and Methods). (GIF 245 kb)

10577_2016_9538_MOESM8_ESM.tif (2.1 mb)
High Resolution Image (TIF 2123 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Daniil A. Maksimov
    • 1
  • Petr P. Laktionov
    • 1
  • Stepan N. Belyakin
    • 1
    • 2
    Email author
  1. 1.Institute of Molecular and Cellular Biology SB RASNovosibirskRussia
  2. 2.Novosibirsk State UniversityNovosibirskRussia

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