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Illumina Library Preparation for Sequencing the GC-Rich Fraction of Heterogeneous Genomic DNA.

ABSTRACT: Standard Illumina libraries are biased toward sequences of intermediate GC-content. This results in an underrepresentation of GC-rich regions in sequencing projects of genomes with heterogeneous base composition, such as mammals and birds. We developed a simple, cost-effective protocol to enrich sheared genomic DNA in its GC-rich fraction by subtracting AT-rich DNA. This was achieved by heating DNA up to 90?°C before applying Illumina library preparation. We tested the new approach on chicken DNA and found that heated DNA increased average coverage in the GC-richest chromosomes by a factor up to six. Using a Taq polymerase supposedly appropriate for PCR amplification of GC-rich sequences had a much weaker effect. Our protocol should greatly facilitate sequencing and resequencing of the GC-richest regions of heterogeneous genomes, in combination with standard short-read and long-read technologies.

SUBMITTER: Tilak MK

PROVIDER: S-EPMC5808798 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Illumina Library Preparation for Sequencing the GC-Rich Fraction of Heterogeneous Genomic DNA.

Tilak Marie-Ka MK Botero-Castro Fidel F Galtier Nicolas N Nabholz Benoit B

Genome biology and evolution 20180201 2

Standard Illumina libraries are biased toward sequences of intermediate GC-content. This results in an underrepresentation of GC-rich regions in sequencing projects of genomes with heterogeneous base composition, such as mammals and birds. We developed a simple, cost-effective protocol to enrich sheared genomic DNA in its GC-rich fraction by subtracting AT-rich DNA. This was achieved by heating DNA up to 90 °C before applying Illumina library preparation. We tested the new approach on chicken DN ...[more]

PMID: 29385572

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Project description:BackgroundMassively parallel sequencing technology is revolutionizing approaches to genomic and genetic research. Since its advent, the scale and efficiency of Next-Generation Sequencing (NGS) has rapidly improved. In spite of this success, sequencing genomes or genomic regions with extremely biased base composition is still a great challenge to the currently available NGS platforms. The genomes of some important pathogenic organisms like Plasmodium falciparum (high AT content) and Mycobacterium tuberculosis (high GC content) display extremes of base composition. The standard library preparation procedures that employ PCR amplification have been shown to cause uneven read coverage particularly across AT and GC rich regions, leading to problems in genome assembly and variation analyses. Alternative library-preparation approaches that omit PCR amplification require large quantities of starting material and hence are not suitable for small amounts of DNA/RNA such as those from clinical isolates. We have developed and optimized library-preparation procedures suitable for low quantity starting material and tolerant to extremely high AT content sequences.ResultsWe have used our optimized conditions in parallel with standard methods to prepare Illumina sequencing libraries from a non-clinical and a clinical isolate (containing ~53% host contamination). By analyzing and comparing the quality of sequence data generated, we show that our optimized conditions that involve a PCR additive (TMAC), produces amplified libraries with improved coverage of extremely AT-rich regions and reduced bias toward GC neutral templates.ConclusionWe have developed a robust and optimized Next-Generation Sequencing library amplification method suitable for extremely AT-rich genomes. The new amplification conditions significantly reduce bias and retain the complexity of either extremes of base composition. This development will greatly benefit sequencing clinical samples that often require amplification due to low mass of DNA starting material.

Project description:Objective: RNA extraction and library preparation from formalin-fixed, paraffin-embedded (FFPE) samples are crucial pre-analytical steps towards achieving optimal downstream RNA sequencing (RNASeq) results. In this study, we assessed 2 Illumina library preparation methods for RNA-Seq analysis using archived FFPE samples from human cancer indications at 2 independent vendors. Methods: Twenty-five FFPE samples from 5 indications (non-small cell lung cancer, colorectal cancer, renal carcinoma, breast cancer, and hepatocellular carcinoma) were included, covering a wide range of sample storage durations (3-25 years-old), sample qualities, and specimen types (resection vs core needle biopsy). Each sample was processed independently by both vendors. Total RNA was isolated using the Qiagen miRNeasy FFPE kit followed by library construction using either TruSeq Stranded Total RNA library preparation kit with Ribo-Zero Gold, or TruSeq RNA Access library preparation kit. Libraries were normalized to 20 pM and sequenced on an Illumina HiSeq 2500 using V3 chemistry in paired-end mode with a read length of 2 × 50 bp. The data were processed through a standard RNASeq pipeline to produce counts and transcripts per millions for each gene in each sample to compare 2 library kits at 2 different vendors. Results: Our data showed that TruSeq RNA Access libraries yield over 80% exonic reads across different quality samples, indicating higher selectivity of the exome pull down by the capture approach compared to the random priming of the TruSeq Stranded Total kit. The overall QC data for FFPE RNA extraction, library preparation, and sequencing generated by the 2 vendors are comparable, and downstream gene expression quantification results show high concordance as well. With the TruSeq Stranded Total kit, the mean Spearman correlation between vendors was 0.87 and the mean Pearson correlation was 0.76. With the TruSeq RNA Access kit, the mean Spearman correlation between vendors was 0.89 and the mean Pearson correlation was 0.73. Interestingly, examination of the cross-vendor correlations compared to various common QC statistics suggested that library concentration is better correlated with consistency between vendors than is the RNA quantity. Conclusions: Our analyses provide evidence to guide selection of sequencing methods for FFPE samples in which the sample quality may be severely compromised.

Dataset Information

Illumina Library Preparation for Sequencing the GC-Rich Fraction of Heterogeneous Genomic DNA.

Publications

Illumina Library Preparation for Sequencing the GC-Rich Fraction of Heterogeneous Genomic DNA.

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