Can you sequence cdna

In this case, ampicillin-resistant cells constitute a good cDNA library, ready for screening. This typically begins preparing multiple replica filters like the one above. Remember, these filters are replicas of bacterial cells containing recombinant plasmids that grow on ampicillin but not streptomycin.

The number of replica filters that must be screened can be calculated from assumptions and formulas for estimating how many colonies must be screened to represent an entire transcriptome i. Once the requisite number of replica filters are made, they are subjected to in situ lysis to disrupt cell walls and membranes.

The result is that the cell contents are released and the DNA is denatured i. The DNA then adheres to the filter in place in situ , where the colonies were. The result of in situ lysis is a filter with faint traces of the original colony below.

Next, a molecular probe is used to identify DNA containing the sequence of interest. The probe is often a synthetic oligonucleotide whose sequence was inferred from known amino acid sequences. These oligonucleotides are made radioactive and placed in a bag with the filter s. DNA from cells that contained recombinant plasmids with a cDNA of interest will bind the complementary probe. The results of in situ lysis and hybridization of a radioactive probe to a replica filter are shown below.

The filters are rinsed to remove un-bound radioactive oligomer probe, and then placed on X-ray film. After a period of exposure, the film is developed. Black spots will form on the film from radioactive exposure, creating an autoradiograph of the filter. The black spots in the autoradiograph correspond to colonies on a filter that contain a recombinant plasmid with your target cDNA sequence below.

Once a positive clone is identified on the film, the corresponding recombinant colony is located on the original plate. This colony is grown up in a liquid culture and the plasmid DNA is isolated. At that point, the cloned plasmid DNA can be sequenced and the amino acid sequence encoded by its cDNA can be inferred from the genetic code dictionary to verify that the cDNA insert in fact encodes the protein of interest.

Once verified as the sequence of interest, a cloned plasmid cDNA can be made radioactive or fluorescent, and used to. Isolated plasmid cDNAs can even be expressed in suitable cells to make the encoded protein. These days, diabetics no longer receive pig insulin, but get synthetic human insulin human made from expressed human cDNAs. Moreover, while the introduction of the polymerase chain reaction PCR , see below has superseded some uses of cDNAs, they still play a role in genome-level and transcriptome-level studies.

The tube full of transformed cells is the cDNA Library. Once verified as the sequence of interest, a cloned plasmid cDNA can be made radioactive or fluorescent, and used to probe for the genes from which they originated. For example, de novo assembly algorithms were so conservative to avoid misassemblies that contigs were often too fragmented. Indeed, after aligning disjoint contigs against the reference genome, we found many contigs overlapping on the genome actually, allowing us to merge those overlapping contigs reliably with the help of the reference genome.

Care had to be taken to use reference assembly because it suffered from two drawbacks. The first concern was locally-intensive e. Since the strain of cDNA libraries is often different from that of the reference genome, alignment of reads against the reference genome should allow locally-intensive polymorphisms to pursue the continuity of the assembled sequences of full-length cDNA clones, but it is almost prohibitive because of the low specificity of such alignment.

The second issue was how to detect exons shorter than the read length of Illumina GA. In reference assembly, spliced alignment was required to find exons shorter than 36 bp the read length , but splicing short reads into two parts deteriorated the alignment specificity significantly, leading to substantial false-positives.

We concluded that reference assembly alone was likely to cause incorrect reconstruction of full-length cDNA sequences and produce many false-positive alignments. Meanwhile, de novo assembly does not suffer from the problem of missing short exons because it assembles the reads directly into the cDNA contigs without alignment to the reference genome.

In our hybrid approach, initial contigs are created using an externally available de novo assembler, and then they are improved using the reference assembly approach Fig. Disjoint de novo contigs that were overlapping on the reference genome in reality were detected and merged by alignment of the de novo contigs with the reference genome.

Some partial de novo assembly could be fixed by alignment of the shotgun short reads to the reference genome. We prepared three input clone sets of full-length cDNA clones to accomplish the following: test the feasibility of shotgun sequencing cDNA clones using Illumina GA; investigate how multiplicity the number of full-length cDNA clones in a clone set affects the quality of the result, and examine whether the effectiveness and accuracy of our approach depends on a particular species.

Each full-length cDNA clone set was shotgun sequenced individually using one lane of a flow cell. To test the feasibility of shotgun sequencing cDNA clones, we randomly chose non-overlapping clones from human full-length cDNA clones [33] whose complete nucleotide sequences had been sequenced independently by the conventional primer-walking method using Sanger sequencers.

Non-overlapping here refers to the criterion that no two clones shared the same genomic regions, which can be easily confirmed by aligning the finished full-length cDNA sequence against the reference genome. S1A shows the length distribution of these cDNA sequences in library 1, which averaged 2, nt in size. One of the clones was found to be chimeric by its alignment to the reference genome, and was therefore excluded from later analyses.

When we selected the clones, we did not check whether the clones would share similar nucleotide sequences e. It contained all the full-length cDNA clones in library 1 and an additional set of full-length cDNA clones to reduce the sequence coverage for the shared full-length cDNA clones. The additional full-length cDNA clones were chosen from existing human full-length cDNA clones [48] while avoiding any pair of clones that suggests the same transcript.

The full nucleotide sequences of the additional full-length cDNA clones are unknown. As the two libraries shared the full-length cDNA clones, the reproducibility of our approach could be also validated by looking to what extent the shared cDNA clones were reconstructed similarly across the libraries. To examine whether the effectiveness and accuracy of our approach depends on a particular species, we prepared library 3, a collection of cDNAs from Toxoplasma gondii.

The human genome is one of the most reliable genome sequences in terms of base quality and continuity, whereas newly analyzed genomes may not be of the same quality. Therefore, measuring the full-length cDNA reconstruction accuracy for non-human species is of great interest to suggest that our approach is applicable to a broad range of species. We expected polymorphisms of higher rate between the reference genome and the shotgun reads, and therefore the clone set was suitable for testing tolerance to polymorphisms.

As a validation full-length cDNA clone set, clones in library 3 were manually finished by the conventional primer-walking using Sanger sequencers see Fig. S1B for the size distribution. Figure 1 illustrates the whole process of our hybrid assembly approach. The detail of amplification of full-length cDNA clones and subsequent sequencing is described in Methods.

One lane was used for each full-length cDNA clone set. The number of obtained raw reads, which were 36 bp in length, ranged from 4. Reads with any ambiguous bases e. For de novo assembly, purity filter was applied using default settings of Solexa pipeline, whereas the reference assembly process utilized non-filtered reads to obtain more aligned reads. After millions of short shotgun reads were obtained, an external assembler was used to produce initial contigs. We used Velvet [47] as the external assembler because it gave the best continuity among tested de novo assemblers see Text S1 , though any other improved assembler can be used instead in the future.

As our hybrid assembly greatly relies on the quality of initial contigs produced by de novo assembly, a good parameter selection for Velvet is a major determinant in our approach. Among a variety of parameters, hash length and read length, are particularly important, and therefore, Velvet was run with hash length ranging from 19 bp to 29 bp and read length from 26 bp to 36 bp, after which the initial contig set was selected among the assemblies with different read length and hash length parameters.

We found that the N50 contig lengths produced in de novo assemblies were highly correlated with the accuracy of the assembly result produced by MuSICA 2 Fig. S2 ; hence we used the Velvet assembly with the best N50 contig length. The alignment with the best alignment score was kept for each contig. When multiple hits tied in alignment score, they were all discarded as repetitive hits. The benefit of this contig improvement by reference alignment over de novo assembly is four fold: a de novo assemblers often yield short contigs due to its inherent difficulty, but reference alignment allows us to merge contigs according to their ranges on the genome sequence; b not all of the contigs are correctly assembled, but misassembled contigs are corrected simply by discarding unaligned parts; c even repetitive short mer regions can be reliably covered when a contig that covers them contains at least one unique region that aligns to only one location on the genome; d contigs are usually longer than reads, so that spliced alignment often retains sufficient specificity even when the length of exons are shorter than reads.

After merging overlapping contigs, the merged contigs do not always represent complete full-length cDNA sequences presumably because those contigs are fragmented due to lack of sequence coverage. To check this hypothetical reason, we aligned raw shotgun reads against the finished full-length cDNA clones, and we found that entire clones were usually covered by the raw shotgun reads, with the exception that regions towards the ends of the clones were underrepresented in the shotgun reads see Supporting Information.

Some parts of exons or intron were not covered by any de novo contigs presumably because short read de novo assemblers discard contigs shorter than a threshold. If two contigs aligned adjacent on the genome are within 1, bp, which is the user-configurable parameter, the gap between them is considered for exon filling and is called an exon gap candidate. When any single nucleotide in an exon gap candidate is not covered by any raw read, the exon gap candidate is not filled.

To avoid filling repetitive regions with spurious matches, the gap is filled only when the number of unique alignments per base is greater than or equal to reads. After exon filling, MuSICA 2 tries to compensate for missing splicing junctions that de novo contigs fail to cover.

If the distance between two adjacent contigs aligned on the reference genome is within to , the gap between them is considered as an intron gap candidate. Raw reads are split into two parts, and then they are aligned against regions near both ends of the intron gap candidates to find actual introns for spliced alignment, see Methods. At this stage, exons are expected to be nearly perfect.

Therefore, the total length of the alignment targets is at least four orders of magnitudes shorter than the whole genome in the case of human Table S2. The narrow alignment targets enabled more reads to be aligned uniquely.

Associating individual full-length cDNA clones with the output contigs is a crucial step to identify the clone material corresponding to any user-requested contig for wet-lab experiments. To this end, we collected Sanger reads from both ends of the full-length cDNA clones as input, though they were not incorporated into the contig generation except for cases explained below. A Sanger read from either end of a clone will be sufficient for clone name assignment.

The filtered alignments usually associated each clone with one contig. Conflicting alignments were resolved on a best-match basis when a single clone could be associated with multiple contigs aligned on totally different locations on the genome. This occurs when the sequence coverage near the ends of the clone was so low that the contig was truncated.

Library 1 consisted of reference human full-length cDNA clones whose complete nucleotide sequences were previously finished by the conventional primer-walking method using Sanger sequencers. We compared the assembly results with those finished sequences to evaluate our hybrid assembly approach.

We aligned the finished sequences against the reference genome to determine their exon-intron structure using BLAT. The alignment results of the finished sequences by the conventional primer-walking method were examined and corrected manually if needed see Text S1.

This structure was used as a reference exon-intron structure. This is because the standard Illumina nebulization protocol is inefficient in shearing DNA molecules near the ends see Text S1. Obviously, using non-PCR methods such as plasmid extraction or TempliPhi for clone amplification would alleviate the problem because they virtually eliminate all clone ends. Although we are improving the experimental protocol, we describe the performance with the original protocol in this paper.

Taking this into account, we instead focused on the consistency of the coding sequence CDS structure, which is a set of genomic regions that correspond to the CDS. The CDS structure comprises of a series of exon coordinates on the reference genome, and does not include any nucleotide sequences. Of all reference full-length cDNA clones, we observed that clones in library 1 had at least one associated contig, whereas the other clones were not associated with any contig.

Table 2 compared the output cDNA sequences and the reference cDNAs in terms of the consistency of the CDS structure, which was obtained by alignment against the reference genome. Comparing with other approaches, MuSICA 2 produced more consistent outputs than de novo -based approaches improved by the use of Sanger reads Fig.

The reconstructed full-length cDNA is consistent with the reference gene if the exon-intron structure of the true CDS was completely contained in the exon-intron structure of the reference gene.

Inconsistency was caused by different exon boundaries, sequence gaps or missing links between exons i. Note that in the case 3 we cannot exclude a possibility that some exon s between them is missing; therefore, the output transcript needs manual finishing in such a case. Since the CDS structure analysis does not evaluate the nucleotide accuracy, we aligned the assembly results against the reference full-length cDNA clones to evaluate the base accuracy of the assembly results Table 3.

The match ratio was over These results suggest that the reconstructed cDNA sequences were reliable enough to annotate CDSs on the reference genome. The accuracy evaluation for the clones library 2 that were not shared with library 1 was difficult. For example, their full-sequences are not available and therefore the correct CDSs are unknown. Sequence coverage across clones is a factor that may affect the sensitivity and accuracy of assembly methods considerably.

To assess the relationship between the accuracy of the MuSICA 2 assembly output and the sequence coverage, we produced simulated shotgun reads of reduced sequence coverage by random sampling of the raw reads. This observation is somewhat different from our expectation, and we analyzed this more precisely. We found that the sequence coverage for individual clones varied widely, and therefore high e. Indeed, Figure 4 shows a clear tendency that clones of low per-clone sequence coverage were often not reconstructed correctly.

To illustrate this more clearly, we binned the clones in all the simulated datasets by their per-clone sequence coverage Fig. This suggests that our hybrid assembly approach will work with higher multiplicity and can reduce sequencing costs further if more uniform sequence coverage across clones is achieved. First, we aligned the shotgun reads against the finished reference clone sequences, allowing up to 3 mismatches. Y-axis shows the number of clones. Every clone is colored according to its consistency of the CDS structure; a red bar shows the proportion of the inconsistent clones in that range.

Clones that were not successfully amplified by PCR are not shown in the histogram, as they always had little sequence coverage by definition. Clones in the simulated datasets were binned according to their per-clone sequence coverage. The bins were of every fold. Note that every full-length cDNA clone was counted 10 times as it appeared with 10 different sequence coverage.

We calculated the percentage of clones left Y-axis classified as consistent or inconsistent in terms of CDS structure for each bin. The number of clones in each bin is also shown right Y-axis. We also studied the CDS reconstruction accuracy for non-human species using library 3, which consisted of full-length cDNA clones from Toxoplasma gondii , a human pathogen.

To create the reference CDS structure for accuracy evaluation, we finished full-length cDNA clones in the library by the conventional primer-walking method using Sanger sequencers. S7 ; those clones were used as a reference in the subsequent evaluation of the CDS structure consistency.

This figure is similar to that for human full-length cDNAs, demonstrating the independency of our approach with regard to species or the quality of the reference genome sequence.

They are often used for functional analysis of genes. To sequence cDNA clones, several methods are proposed but our approach is outstanding in low sequencing cost.

The precise calculation is:. On the other hand, previous sequencing approaches using Sanger methods are obviously much more costly. For example, Hokkaido System Science Co. Multiclone shotgun sequencing using the Sanger method is cheaper than primer-walking but still much more expensive than our method. Assuming 6. Second-generation sequencers deliver DNA sequencing at a lower cost.

For example, Salehi-Ashtiani et al. This approach also provides a fast way for sequencing full-length cDNA clones but still more expensive than our method. Since our approach has significantly reduced sequencing cost, MuSICA 2 accelerates numerous full-length cDNA sequencing projects for a variety of species, and will provide more accurate knowledge about transcriptome complexity. The collections of full-length cDNA clones are also useful for experimental analysis to reveal the biological functions of proteins.

For example, recombinant proteins can be produced using expression vectors made from individual full-length cDNA clones with the complete sequences of their CDSs. Therefore, we contribute a new method for transcriptome studies that simplifies the task of assembling targeted isoforms. Although the requirement of having Sanger reads adds extra costs beyond that of the Illumina GA sequencing reagents, to the best of our knowledge, there is no cheaper method that allows one to determine the correspondence between reconstructed cDNA sequences and individual clones, suggesting that multi-clone shotgun sequencing using the Illumina GA with additional Sanger reads is the most cost-effective method for obtaining complete sequences of the CDSs associated with individual cDNA clones.

With Sanger reads, clones that are not amplified by PCR can be easily detected and then we can sequence them in another run. Our approach requires that no two full-length cDNA clones in a same library should overlap on the reference genome because of the difficulty in reconstruction of individual full-length cDNA clones overlapping on the reference genome.

If sequencing centers have full-length cDNA clones from a variety of distantly related species, they can distribute them over flowcell lanes to increase throughput while keeping the accuracy, because clones from distantly related species do not interfere much with one another. Second-generation sequencers perform full-length cDNA sequencing an order of magnitude cheaper and faster than conventional full-length cDNA sequencing using the Sanger method.

However, short-read sequencers have higher sequencing error rates than Sanger sequencers and have yet to be characterized regarding sequencing error patterns, and de novo assembly algorithms of short shotgun reads are still under development for general application including de novo sequencing of mammalian genome or genes.

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Reprints and Permissions. Agarwal, S. Nat Commun 6, Download citation. Received : 20 March Accepted : 02 December Published : 21 January Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article.

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Abstract Massively parallel strand-specific sequencing of RNA ssRNA-seq has emerged as a powerful tool for profiling complex transcriptomes. You have full access to this article via your institution. Full size image. Results Generation of the DLAF ssRNA-seq library In a recent systematic and comprehensive comparison of various methods for ssRNA-seq libraries, the dUTP method 8 outperformed other methods in multiple ways, including relative ease in experimentation and computational handling and a higher quality of data Increased library yields The final yield of a library preparation method is an important indicator of its utility, especially when RNA is available only in small amounts.

Increased mappability and higher mapping to unique regions Multiplexed libraries were subjected to either single- or paired-end sequencing on an Illumina HiSeq instrument. Full size table.