PacBio Whole Genome Shotgun Sequences for the D. melanogaster Reference Strain

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Posted 31 Jul 2013 in drosophila, genome bioinformatics, high throughput sequencing

As part of a collaboration with Danny Miller and Scott Hawley from the Stowers Institute, we have generated whole genome shotgun sequences using PacBio RS technology for the Drosophila melanogaster y; cn, bw, sp strain (Bloomington 2057), the same strain that was also used to assemble the D. melanogaster reference genome sequence. We’ve been meaning to release these data to the community since we got the data in April, but have been waylaid by teaching commitments and a spate of recent server problems.  Prompted by Danny’s visit to the Bergman Lab over the last two weeks and the generous release by Dmitri Petrov’s lab of a data set of Illumina long reads using the Moleculo technology for the same strain of D. melanogaster, we’ve finally gotten around to bundling these D. melanogaster PacBio sequences for general release. We’re hoping that the availability of both PacBio and Moleculo long-read data for the same strain that has one of the highest quality reference genomes for any species will allow the genomics community to investigate directly the pros/cons of each of these new exciting technologies.

These PacBio sequence data were generated by the University of Michigan DNA Sequencing Core facility on a PacBio RS using DNA from 10 adult males. Flies were starved for 4 hours to reduce microbial DNA contaminants before freezing at -80oC and prepped using the Qiagen DNeasy Blood & Tissue Kit (catalog number 69504). Six SMRT cells were used for sequencing 5 Kb fragment libraries on two Long Read mode runs: 1 cell on the first run (Run53) and 5 cells on the second run (Run55). Excluding the circular consensus (CCS) reads, and combining data from the S1 and S2 files for each of the six cells, we obtained 1,357,183,439 bp of raw DNA sequence from this experiment or roughly 7.5x coverage of the 180 Mb male D. melanogaster genome.

A ~63 Gb tar.gz of the entire PacBio long read dataset including .fasta, .fastq, and .h5 files can be found here. To help newcomers to PacBio data (like us) get over the hurdle of installing the PacBio long-read aligner blasr, we are also distributing CentOS 6/Scientific Linux 64-bit and source RPMs for blasr, made by Peter Briggs in the University of Manchester Bioinformatics Core Facility.

We have also generated 100bp paired-end Illumina data from the same stock, to aid with error-correction of the PacBio long reads. These data were generated by the Stowers Institute Molecular Biology Core facility. As above, genomic DNA was prepared from 10 starved, adult males using the Qiagen DNeasy Blood & Tissue Kit. 1ug of DNA from each was fragmented using a Covaris S220 sonicator (Covaris Inc.) to 250 base pair (bp) fragments by adjusting the treatment time to 85 seconds. Following manufacturer’s directions, short fragment libraries were made using the KAPA Library Preparation Kits (KAPA Biosystems, Cat. No. KK8201) and Bioo Scientific NEXTflex™ DNA Barcodes (Bioo Scientific, Cat. No. 514104). The resulting libraries were purified using Agencourt AMPure XP system (Beckman Coulter, Cat. No. A63880), then quantified using a Bioanalyzer (Agilent Technologies) and a Qubit Fluorometer (Life Technologies).  The library was pooled with several other strains, re-quantified and run as high output mode on six 100 bp paired-end lanes on an Illumina HiSeq 2000 instrument, using HiSeq Control Software and Real-Time Analysis (RTA) version Secondary Analysis version CASAVA-1.8.2 was run to demultiplex reads and generate FASTQ files.

The Illumina 100 bp paired-end dataset in .fastq format can be at ENA under accession ERX645969. Additional Illumina data for the ISO1 reference strain has been generated previously by Chuck Langley’s lab, either from mixed adult males and females (SRX040484) or haploid embryos (SRX040485, SRX040486, SRX040491) that could be used to supplement the PacBio error correction.

As with previous unpublished data we have released from the Bergman Lab, we have chosen to release these genomic data under a Creative Commons CC-BY license, which requires only that you credit the originators of the work as specified below.  However, we hope that users of these data respect the established model of genomic data release under the Ft. Lauderdale agreement that is traditionally honored for major sequencing centers.  Until the paper describing these genomic data is published, please cite:

  • Miller, D.E., C.B. Smith, R.S. Hawley and C.M. Bergman (2013) PacBio Whole Genome Shotgun Sequences for the D. melanogaster Reference Strain.

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