DNA Library

Example 3

We tested the ability of mouse embryonic stem cells to tolerate s⁴U metabolic RNA-labeling after 12 h or 24 h at varying s⁴U concentrations (FIG. 6A). As reported previously, high concentrations of s⁴U compromised cell viability with an EC₅₀ of 3.1 mM or 380 μM after 12 h or 24 h labeling, respectively (FIG. 6A). Hence, we employed labeling conditions of 100 μM s⁴U, which did not severely affect cell viability. Under these conditions, we detected a steady increase in s⁴U-incorporation in total RNA preparation 3 h, 6 h, 12 h, and 24 h post labeling, as well as a steady decrease 3 h, 6 h, 12 h, and 24 h after uridine chase (FIG. 6B). As expected, the incorporation follows a single exponential kinetics, with a maximum average incorporation of 1.78% s⁴U, corresponding to one s⁴U incorporation in every 56 uridines in total RNA (FIG. 6C). These experiments establish s⁴U-labeling conditions in mES cells, which can be employed to measure RNA biogenesis and turnover rates under unperturbed conditions.

To test the ability of the method to uncover s⁴U incorporation events in high throughput sequencing datasets we generated mRNA 3′ end libraries (employing Lexogen's QuantSeq, 3′ mRNA-sequencing library preparation kit) using total RNA prepared from cultured cells following s⁴U-metabolic RNA labeling for 24 h (FIG. 7) (Moll et al., supra). Quant-seq 3′ mRNA-Seq Library Prep Kit generates Illumina-compatible libraries of the sequences close to the 3′end of the polyadenylated RNA, as exemplified for the gene Trim28 (FIG. 8A). In contrast to other mRNA-sequencing protocols, only one fragment per transcript is generated and therefore no normalization of reads to gene length is needed. This results in accurate gene expression values with high strand-specificity.

Furthermore, sequencing-ready libraries can be generated within only 4.5 h, with ˜2 h hands-on time. When combined with the invention, Quant-seq facilitates the accurate determination of mutation rates across transcript-specific regions because libraries exhibit a low degree of sequence-heterogeneity. Indeed, upon generating libraries of U-modified RNA through the Quant-seq protocol from total RNA of mES cells 24 h after s⁴U metabolic labeling we observed a strong accumulation of T>C conversions when compared to libraries prepared from total RNA of unlabeled mES cells (FIG. 8B). In order to confirm this observation transcriptome-wide, we aligned reads to annotated 3′ UTRs and inspected the occurrence of any given mutation per UTR (FIG. 9). In the absence of s⁴U metabolic labeling, we observed a median mutation rate of 0.1% or less for any given mutation, a rate that is consistent with Illumina-reported sequencing error rates. After 24 h of s⁴U metabolic labeling, we observed a statistically significant (p<10⁻⁴, Mann-Whitney test), 25-fold increase in T>C mutation rates, while all other mutations rates remained below expected sequencing error rates (FIG. 9). More specifically, we measured a median s⁴U-incorporation of 2.56% after 24 h labeling, corresponding to one s⁴U incorporation in every 39 uridines. (Note, that median incorporation frequency for mRNA are higher than estimated by HPLC in total RNA [FIG. 6C], most certainly because stable non-coding RNA species, such as rRNA, are strongly overrepresented in total RNA.) These analyses confirm that the new method uncovers s⁴U-incorporation events in mRNA following s⁴U-metabolic RNA labeling in cultured cells.

We expect the same incorporation results of other modified nucleotides, such as s⁶G or 5-ethynyluridine, as reported previously (Eidinoff et al., Science. 129, 1550-1551 (1959); Jao et al. PNAS 105, 15779-15784 (2008); Melvin et al. Eur. J. Biochem. 92, 373-379 (1978); Woodford et al. Anal. Biochem. 171, 166-172 (1988)).

Example 4

FIGS. 5A-B show an exemplary nucleic acid library method to reverse the orientation of an analyte sequence in a member of a nucleic acid library. FIG. 5A shows an exemplary member of a nucleic acid library including, in a 5′ to 3′ direction, a ligation sequence, a barcode (e.g., a spatial barcode or a cell barcode), unique molecular identifier, a reverse complement of a first adaptor, an amplification domain, a capture domain, a sequence complementary to an analyte, and a second adapter.

The ends of the double-stranded nucleic acid can be ligated together via a ligation reaction where the ligation sequence splints the ligation to generate a circularized double-stranded nucleic acid also shown in FIG. 5A.

The circularized double-stranded nucleic acid can be amplified to generate a linearized double-stranded nucleic acid product, where the orientation of the analyte is reversed such that the 5′ sequence (e.g., 5′ UTR) is brought in closer proximity to the barcode (e.g., a spatial barcode or a cell barcode) (FIG. 5B). The first primer includes a sequence substantially complementary to the reverse complement of the first adaptor and a functional domain. The functional domain can be a sequencer specific flow cell attachment sequence (e.g., P5). The second primer includes a sequence substantially complementary to the amplification domain.

The resulting double-stranded member of the nucleic acid library including a reversed analyte sequence (e.g., the 5′ end of the analyte sequence is brought in closer proximity to the barcode) can undergo standard library preparation methods, such as library preparation methods used in single-cell or spatial analyses. For example, the double-stranded member of the nucleic acid library lacking all, or a portion of, the sequence encoding the constant region of the analyte can be fragmented, followed by end repair, A-tailing, adaptor ligation, and/or amplification (e.g., PCR) (FIG. 5C). The fragments can then be sequenced using, for example, paired-end sequencing using TruSeq Read 1 and TruSeq Read 2 as sequencing primer sites, or any other sequencing method described herein.

As a result of the methods described in this Example, sequences from the 5′ end of an analyte will be included in sequencing libraries (e.g., paired end sequencing libraries). Any type of analyte sequence in a nucleic acid library can be prepared by the methods described in this Example (e.g., reversed).

EXAMPLE 36

Peripheral blood mononuclear cells were prepared from blood samples obtained from llama No. 45 and No. 46 using Ficoll-Hypaque according to the manufacturer's instructions. Next, total RNA extracted was extracted from these cells and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage was prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein) and stored after filter sterilization at 4° C. for further use.