Inexpensive multiplexed library preparation for megabase-sized genomes

Abstract

Whole-genome sequencing has become an indispensible tool of modern biology. However, the cost of sample preparation relative to the cost of sequencing remains high, especially for small genomes where the former is dominant. Here we present a protocol for rapid and inexpensive preparation of hundreds of multiplexed genomic libraries for Illumina sequencing. By carrying out the Nextera tagmentation reaction in small volumes, replacing costly reagents with cheaper equivalents, and omitting unnecessary steps, we achieve a cost of library preparation of $8 per sample, approximately 6 times cheaper than the standard Nextera XT protocol. Furthermore, our procedure takes less than 5 hours for 96 samples. Several hundred samples can then be pooled on the same HiSeq lane via custom barcodes. Our method will be useful for re-sequencing of microbial or viral genomes, including those from evolution experiments, genetic screens, and environmental samples, as well as for other sequencing applications including large amplicon, open chromosome, artificial chromosomes, and RNA sequencing.

Fig 1. Schematic of library preparation workflow.

Fig 2. Dependence of fragment size distribution on input gDNA concentration and bead volume.

DNA fragment size distribution is affected by starting genomic DNA concentration (rows) as described in Module 1 as well as the relative amount of bead buffer used in PCR clean-up (columns) as described in Module 4. Size distribution is measured by BioAnalyzer and reported in fluorescence units. Data is from Stenotrophomonas maltophilia (67% GC, 4.8 Mb genome). At high initial gDNA concentration (1.25 ng/μl) the fragment distribution is right-skewed, though this anomalous peak does not appear to significantly affect sequencing output.

Fig 3. Distribution of the fraction of unique reads over 261 E. coli samples.

Samples had between 0.2 and 2.8 million reads with 90% of samples having over 1 million reads. Raw reads were filtered and then aligned to a reference genome using bowtie2. Unique reads are those that appear only once in the alignment for a particular sample. These are the reads that remain after use of the rmdup tool in samtools. Non-unique reads arise primarily when the same tagmented fragment is amplified during PCR. A low fraction of non-unique reads implies a diversity of fragments after tagmentation, and that errors introduced during PCR will not reach high frequencies.

Fig 4. Correspondence between BioAnalyzer traces and the length distribution of aligned reads.

Panels A-C show three representative BioAnalyzer traces from three sample preparations of S. maltophilia. Panels D-F show the corresponding estimated fragment-size distributions (black) and the actual distributions of fragment lengths imputed from alignment to the reference genome (blue). A BioAnalyzer trace f(x) shows fluorescence f at fragment length x. However, we are interested in n(x), the (relative) number of fragments n of length x. Since fluorescence of a DNA fragment is proportional to its length, n(x) ∝ f(x) / x. Note that sequencing can be successful despite the presence of apparently very long fragments (which are likely heteroduplexes) in the BioAnalyzer traces (Panels C and F).

Fig 5. Size and number of aligned fragments as a function of post-PCR DNA concentration.

Data is from two plates of E. coli samples, with 83 and 95 samples per plate (S2 Table). Input gDNA concentrations ranged from 2 to 25ng/μl, and were standardized to 0.5ng/μl. Based on estimated fragment-length distributions, Plates 1 and 2 were pooled in mass ratio 0.8:1. In this preparation, 2 samples (1%) failed to yield libraries, and 17 (10%) produced low, but usable, numbers of reads (between 0.1 and 0.4 million)