Information

Sequencing rtPCR product


So I have a validated set of primers for rtPCR from Biorad that contains SYBR green. If I do rtPCR, can I use the rtPCR product after purifying it with a Qiagen PCR purification kit? Also, I'm under the impression that the sequencing company needs primers to do sequencing, do I just use the PCR primer or one of the "universal" primers the company has?


You can use the PCR purification kit for cleaning up the DNA.

You can use the primers that you used for RTPCR for sequencing. The universal primers would work only if the sequence complementary to it is there in the 3' end of your amplicon.


Sequencing RT-PCR product - (Mar/14/2013 )

So I have a validated set of primers for rtPCR from Biorad that contains SYBR green. If I do rtPCR, can I use the rtPCR product after purifying it with a Qiagen PCR purification kit? Also, I'm under the impression that the sequencing company needs primers to do sequencing, do I just use the PCR primer or one of the "universal" primers the company has?

By rtPCR you mean real time- or reverse transcription- PCR? If the former, to avoid confusion it is better to use quantitative PCR (qPCR).

As far as I know primers do not contain sybr green, the sybr agent is much like ethidium bromide in that it relies on intercalation between the DNA strands at a particular density per bp, and as such sybr is added as part of the PCR mastermix. It is possible however to get primers that are fluorescently (fl) tagged such as Taqman assay primers.

In practice it shouldn't matter whether the primers are tagged for use in sequencing, though I have never tried it. I can imagine that a Fl tagged primer may obscure the signal from some of the initial base calls if used though.

For sequencing, yes the company does require primers, you need to set up a tube that contains the target DNA and ONE primer only - if you have both primers in the same tube you will get mixed signal as the forward and reverse regions get sequenced simultaneously (think about how DNA is replicated and the orientation of the two strands).

The universal primers are only for sequencing off plasmids (and even then, only for some plasmids) if your product is not contained in a plasmid you need to supply the correct primer.

bob1 on Fri Mar 15 04:11:59 2013 said:

By rtPCR you mean real time- or reverse transcription- PCR? If the former, to avoid confusion it is better to use quantitative PCR (qPCR).

As far as I know primers do not contain sybr green, the sybr agent is much like ethidium bromide in that it relies on intercalation between the DNA strands at a particular density per bp, and as such sybr is added as part of the PCR mastermix. It is possible however to get primers that are fluorescently (fl) tagged such as Taqman assay primers.

In practice it shouldn't matter whether the primers are tagged for use in sequencing, though I have never tried it. I can imagine that a Fl tagged primer may obscure the signal from some of the initial base calls if used though.

For sequencing, yes the company does require primers, you need to set up a tube that contains the target DNA and ONE primer only - if you have both primers in the same tube you will get mixed signal as the forward and reverse regions get sequenced simultaneously (think about how DNA is replicated and the orientation of the two strands).

The universal primers are only for sequencing off plasmids (and even then, only for some plasmids) if your product is not contained in a plasmid you need to supply the correct primer.


Thanks for clearing this up for me. Yes I meant qPCR. And no the primers don't contain SYBR green, which is part of the master mix, you're right. Made some errors describing what I want to do because this is the first time I've ever tried PCR.

You should ask the company whether the SYBR die interferes with their sequencers. The safest way to do so would be purifying the product threw the column (i have used ones from Thermo Fisher - GenJet PCR purification kit) - id does what it says


Direct Sequencing of PCR Products

To obtain high quality sequencing data, it is very important that the PCR reaction is specific and strong. If the PCR product is a smear on an agarose gel, or more than one band is present, the likelihood of obtaining good sequence data is low.

You must remove all PCR primers and unincorporated nucleotides before the product is sequenced. Sequencing uses only one primer instead of the two used in PCR. If you do not remove both primers, you will get two sequences superimposed on each other that are not readable.

It is OK to use a PCR primer for sequencing as long as it matches our conditions. Please see Primer Guidelines for more information.

Double check PCR concentration using an analytical agarose gel. Over concentrating your template will not give you a better sequence and could potentially interfere with neighboring researchers samples on the instrument.

Applied Biosystems has a useful manual for PCR Sequencing which can be downloaded as a PDF file. You will need Acrobat Reader to complete the download.

Another useful booklet is the Qiagen Guide to Template Purification and DNA Sequencing.

The recommendations for technical booklets given above in no way represent an endorsement of either ABI or Qiagen products.


Universal primer set for amplification and sequencing of HA0 cleavage sites of all influenza A viruses

Sequence analysis of the endoproteolytic cleavage site within the hemagglutinin (HA) precursor protein HA(0) is fundamental for studies of the molecular biology of influenza A viruses, in particular, for molecular pathotyping of subtype H5 and H7 isolates. A current problem for routine diagnostics is the emergence of new strains of the H5 or H7 subtype or even other subtypes which escape detection by commonly used reverse transcription-PCR (RT-PCR) protocols. Here, the first pan-HA (PanHA) RT-PCR assay targeting the HA(0) cleavage site of influenza A viruses of all 16 HA subtypes is reported. The assay was assessed in comparison to H5 and H7 subtype-specific RT-PCRs for the HA(0) cleavage site and a real-time RT-PCR detecting the M gene. A panel of 92 influenza A viruses was used for validation. Sequence data for influenza A viruses from 32 allantoic fluid samples and 11 diagnostic swab samples of all 16 HA subtypes were generated by direct sequencing of the PanHA RT-PCR products. The results demonstrate that the new PanHA RT-PCR assay--followed by cycle sequencing--can complement existing methods and strengthen the reliability of influenza A virus diagnostics, allowing both molecular pathotyping (H5 and H7) and subtyping (non-H5 or -H7) within a single approach.


RESULTS AND DISCUSSION

By computational RNomics strategy, the students have identified about 80 snoRNAs with structural features typical of the box C/D snoRNA family from N. crassa, C. glabrata, D. hansenii, K. lactis, and Y. lipolytica. Computational analysis showed that these snoRNAs exist in single or gene cluster form and show diverse genomic organizations in different fungi. Most snoRNAs from C. glabrata and K. lactis snoRNA are independently transcribed, while snoRNAs from N. crassa, D. hansenii, and Y. lipolytica were nested within introns of host genes.

In the laboratory, students functioned individually. Each student selected one snoRNA or one snoRNA cluster to complete his experiment. At first, the students isolated total RNA from these fungi. Using the method mentioned above, all students obtained 250–300 μg of total RNA from 10 ml of fungal cell culture. The A260/A280 ratios of the RNA preparations were 1.85–1.98, illustrating good quality RNA with respect to purity. In addition, the agarose formaldehyde denaturing gel electrophoresis showed three distinct bands and the 25S rRNA band was significantly more intense than the 18S rRNA band, indicating that the RNAs extracted from fungi were fully intact and RNA degradation did not occur. The results of two student's gels are shown in Fig. 1.

Electrophoresis analysis of total RNA from fungi. Lane 1, total RNA from N. crassa. Lane 2, total RNA from D. hansenii. rRNAs of 25S, 16S, and 5.8S are highly visible on the gel.

Then, the students carried out RT-PCR to analyze the expression of snoRNA. Most students obtained RT-PCR products. For students who were not able to obtain RT-PCR products, the instructors had students who were successful to share their products so that all students could continue to progress through the project. Figure 2 shows one student's results. The student analyzed a snoRNA gene cluster located in an antisense strain of a putative protein gene in N. crassa genome by RT-PCR. Since the gene contains two introns (Fig. 2a) according to analysis software, the RT-PCR products should contain three bands. As expected, RT-PCR products from N. crassa total RNA, with P1/P2 primer pair, were composed of three bands, the largest (511 bp) of which matched the sequences of the PCR product from N. crassa genomic DNA, the smallest RT-PCR product (120 bp) corresponded to a spliced transcript that lacks two intronic sequences, and the middle one was the splice intermediate, in which one intron has been removed (Fig. 2b). Cloning and sequencing of the three RT-PCR products verified the occurrence of mature spliced transcript and splice intermediates from which the introns have been removed at the exact position as predicted (Fig. 3). The results above showed that the computational analysis of splice sites is correct and the RNA prepared by the student is fully intact with which all expressed products, including precursors, splice intermediates, and mature spliced transcript of a gene, can be obtained.

snoRNA gene cluster flanking sequence and RT-PCR analyses. (a) snR55 and snR61 flanking sequence from N. crassa. Coding regions for snoRNAs are shaded, the exons of the noncoding RNA are in capital letters intron is in lowercase letter the splice sites and donor/acceptor sequences (boxed) are indicated. Arrows mark the locations of the primers used for RT-PCR analysis. (b) RT-PCR analysis. Lane 1, RT-PCR, after reverse transcription of N. crassa total RNA with primer P1, PCR was performed with primer pair P1/P2. The small RT-PCR product (120 bp) corresponds to the spliced transcript unspliced precursor of transcript (511 bp) is also detectable. Lane 2, DNA control, PCR of N. crassa genomic DNA with primer pair P1/P2 predicted PCR product is 511 bp. Lane 3, PCR control, PCR of N. crassa total RNA with primer pair P1/P2.

The sequence of the RT-PCR product corresponds to the spliced transcript from the snoRNA gene cluster. The position of the removed intron is denoted by arrowhead. (a) Detection of removal of the first intron from a splice intermediate. (b) Detection of removal of the second introns from a splice intermediate.

In summary, this laboratory procedure represents an effective and consistent method for preparing advanced students to participate in research. This procedure introduces students to the concept of RNomics, noncoding RNA, and RNA splicing, to the application of biological databases and bioinformatics tools, and to the practice of basic molecular techniques. We believe that the procedure should be applicable to study other noncoding RNA without much modification.


Direct sequencing of hepatitis A virus and norovirus RT-PCR products from environmentally contaminated oyster using M13-tailed primers

Human norovirus (HuNoV) and hepatitis A (HAV) are recognized as leading causes of non-bacterial foodborne associated illnesses in the United States. DNA sequencing is generally considered the standard for accurate viral genotyping in support of epidemiological investigations. Due to the genetic diversity of noroviruses (NoV), degenerate primer sets are often used in conventional reverse transcription (RT) PCR and real-time RT-quantitative PCR (RT-qPCR) for the detection of these viruses and cDNA fragments are generally cloned prior to sequencing. HAV detection methods that are sensitive and specific for real-time RT-qPCR yields small fragments sizes of 89-150bp, which can be difficult to sequence. In order to overcome these obstacles, norovirus and HAV primers were tailed with M13 forward and reverse primers. This modification increases the sequenced product size and allows for direct sequencing of the amplicons utilizing complementary M13 primers. HuNoV and HAV cDNA products from environmentally contaminated oysters were analyzed using this method. Alignments of the sequenced samples revealed ≥95% nucleotide identities. Tailing NoV and HAV primers with M13 sequence increases the cDNA product size, offers an alternative to cloning, and allows for rapid, accurate and direct sequencing of cDNA products produced by conventional or real time RT-qPCR assays.


NEBNext ® ARTIC products for SARS-CoV-2 sequencing

Especially with the ongoing emergence of SARS-CoV-2 variants that affect virus transmission and other metrics significant to public health, there is an increasing need for reliable, accurate and fast methods for sequencing SARS-CoV-2. The NEBNext ARTIC kits are based on the original work of the ARTIC Network, who quickly adapted their protocols to SARS-CoV-2 (Josh Quick 2020. nCoV-2019 sequencing protocol v2 (GunIt)).

The ARTIC method is a multiplexed amplicon-based whole-viral-genome sequencing approach (Figure 1), and the NEBNext ARTIC kit options are compatible with Illumina and Oxford Nanopore Technologies sequencing platforms. The two kits compatible with Illumina sequencing generate library inserts of

400 bp, for 2 x 75 or 2 x 250 sequencing, respectively (Figure 2). The NEBNext ARTIC SARS-CoV-2 RT-PCR Module contains only the reagents required for cDNA synthesis and targeted cDNA amplification from SARS-CoV-2 genomic RNA.

Figure 1: NEBNext ARTIC Workflow Overview

For more detailed workflows, use the links below:

Figure 2: NEBNext ARTIC Kit Options



NEBNext ARTIC kits include V3 ARTIC primer pools, which have been balanced using methodology developed at NEB based on empirical data from sequencing. These balanced primers provide greater uniformity of genome coverage (Figure 3) from 10-10,000 SARS-CoV-2 genome copies. The reagents for RT-PCR and library prep are optimized for the SARS-CoV-2 ARTIC workflow.

Figure 3: Fewer reads are required to completely cover the genome with the NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies)

Integrative Genome Viewer visualization of read coverage across the SARS-CoV-2 genome. Amplicons were generated from 1,000 copies of SARS-CoV-2 viral gRNA inputs (ATCC VR-1986 and VR-1991) in 100 ng of Universal Human Reference RNA (ThermoFisher QS0639) using IDT ARTIC nCoV-2019 V3 Panel (&ldquoStandard&rdquo) or the NEBNext balanced ARTIC SARS-CoV-2 primer pools. Libraries were constructed using the NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies) and the Oxford Nanopore Technologies Native Barcoding Expansion kits 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114), Ligation Sequencing Kit (SQK-LSK109) and SFB Expansion Kit (EXP-SFB001). Sequencing was on a GridION instrument using R9.4.1 flow cells. Minimap2 was used with 24500 reads or 250x data for the mapping against SARS-CoV-2 Wuhan-Hu-1.

Figure 4: Genome Coverage at a wide range of input amounts, with the NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina)

Amplicons were generated from 1,000 copies of SARS-CoV-2 viral gRNA inputs (ATCC VR-1986 and VR-1991) in 100 ng of Universal Human Reference RNA (ThermoFisher QS0639) using NEBNext balanced ARTIC SARS-CoV-2 primer pools, with or without NEBNext ARTIC Human Control Primer Pairs. Libraries were constructed using the NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina) and sequenced on a MiSeq instrument (2x75 bp). The fraction of the genome covered at each depth was determined for a range of inputs and reads down-sampled to 10,000, 100,000, 500,000 and 1,000,000.

RT reaction conditions are the same for all input amounts, and for Illumina applications, a novel DNA polymerase formulation for the enrichment of next-generation sequencing libraries eliminates the need to normalize amplicon concentrations prior to library preparation.

  • Improved uniformity of SARS-CoV-2 genome coverage depth
  • Streamlined, high-efficiency protocols
  • Effective with a wide range of viral genome inputs (10-10,000 copies)
  • Available for Illumina and Oxford Nanopore Technologies sequencing platforms
  • Single RT conditions for all input amounts
  • No requirement for amplicon normalization prior to library preparation (Illumina-compatible kits)
  • Optional use control human primers provided
  • Includes NEBNext Sample Purification Beads (SPRIselect ® )
  • Library adaptors and primers available separately

Read what leaders in ARTIC SARS-CoV-2 sequencing are saying

The NEBNext ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies) will provide researchers with the most convenient way to perform nanopore sequencing of SARS-CoV-2. When developing the ARTIC protocol we have focused on recovery of near-complete genomes from challenging clinical samples with <Ct33. We adopted a 400 bp tiling amplicon approach because it provides robust performance across a wide range of inputs including high Ct and degraded RNA samples which are often encountered. The native barcoding workflow provides full-length amplicons compatible with best practice demultiplexing and reference-based consensus building while the PCR-free library preparation minimizes amplicon contamination risk. We have worked in collaboration with NEB in optimizing the protocol to minimize reagent volumes while improving performance and this now also benefits from optimized primer pools which greatly improve genome completeness and reduce read coverage requirements. The compatibility with the 96-barcode native barcoding expansion kit allows the flexibility to run between 24 and 96 samples depending on your requirements.

- Joshua Quick, Ph.D., of the University of Birmingham and the ARTIC Network

We have been impressed by the great performance of the NEBNext ARTIC FS kit in our sequencing of human samples containing SARS-CoV-2 RNA. With this kit we have been successfully processing samples of varying quality and quantity and have been amazed by its sensitivity and remarkable uniformity of the viral genome coverage. The quick turnaround time of the robust workflow has been truly beneficial for us.

- Vladimir Benes, Head of Genomics Core Facility at the European Molecular Biology Laboratory

The NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit ensures rapid, accurate and reliable determination of the SARS-CoV-2 whole genome. Due to its ease of use, the NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit enables rapid integration into the routine NGS workflow. Furthermore, the kit still works robust with difficult clinical samples (ct>30). I would recommend the NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit 100%.


Double stranded DNA templates denature at a temperature that is determined in part by their G+C content. The higher the proportion of G+C, the higher the temperature required to separate the strands of template DNA. The longer the DNA molecules, the greater the time required at the chosen denaturation temperature to separate the two strands completely. If the temperature for denaturation is too low or if the time is too short at AT rich regions of the template DNA will be denatured. When the temperature is reduced later in the PCR cycle, the template DNA will reanneal into fully native condition. In PCRs catalyzed by Taq polymerase, denaturation is carried out at 94-95 o C, which is the highest temperature that the enzyme can endure for 30 or more cycles without sustaining excessive damage. In the first cycle of PCR, denaturation is sometimes carried out for 5 minutes to increase the probability that long molecules of template DNA are fully denatured. However this extended period of denaturation temperature is unnecessary for linear DNA molecules as it may be deleterious sometimes. Denaturation for 45 seconds at 94-95oC is routinely used to amplify linear DNA molecules whose GC content is <55% and higher temperature for template and/or target DNAs whose GC content is >55%. So much more heat tolerant polymerases are preferred in such cases.

The temperature used for the annealing step is critical. If the annealing temperature is too high, the oligonucleotide primers anneal poorly, if at all to the template and the yield of amplified DNA is very low. If the annealing temperature is too low, non specific annealing of primers may occur, resulting in the amplification of unwanted segments of DNA. Annealing is usually carried out 3-5o C lower than the calculated melting temperature at which the oligonucleotide primers dissociate from their templates. Many formulas exist to determine the theoretical Tm, but none of them are accurate for oligonucleotide primers for all lengths and sequences. It is best to optimize the annealing conditions by performing a series of trial PCRs at temperatures ranging from 2C to 10 C below the lower of melting temperatures calculated for the two oligonucleotide primers. Alternatively, the thermal cycler can be programmed to use progressively lower annealing temperatures in consecutive pairs of cycles ("touchdown" PCR. Instead of surveying a variety of annealing conditions in separate PCRs, optimization is achieved by exposing a single PCR to a sequential series of annealing temperatures in successive cycles of the reaction.

Extension of oligonucleotide primers is carried out at or near the optimal temperature for DNA synthesis catalyzed by the thermostable polymerase, which in the case of Taq polymerase is 72-78 o C. In the first two cycles, extension from one primer proceeds beyond the sequence complementary to the binding site of the other primer. In the next cycle, the first molecules are produced whose length is equal to the segment of DNA delimited by the binding sites of the primers. From the third cycle onwards, this segment of DNA is amplified geometrically, whereas longer amplification products accumulate arithmetically. The polymerization rate of Taq polymerase is

2000 nucleotides /minute at the optimal temperature (72-78 o C) and as a rule of thumb, extension is carried out for 1 minute for every 1000bp of product. For the last cycle of PCR, many investigators use an extension time that is 3 times longer than the previous cycles, ostensibly to allow completion of all amplified products.


Introduction

Overview of high-throughput sequencing strategies

The shotgun-based strategy—the technique of preference for sequencing large DNA genomes—has been used for the complete genome characterization of a range of eukaryotes and prokaryotes 1,2,3,4,5,6 . Genomic DNA is sheared into 2–50 kbp fragments 2 , 3′ and 5′ ends are repaired and cloned into a bacterial vector to produce a library. The 700 bp terminal regions of these clones are sequenced with insert-independent vector primers 7 , and the complete sequence is reconstructed from overlapping fragments. Large-scale sequencing projects have spurred on recent technical improvements to reduce the cost and time required to obtain single-sequence resolution. These advances consist mainly of alternatives to creating bacterial libraries and to Sanger sequencing biochemistry 8 . Hence, integrated sequencing procedures that include library production, DNA amplification, sequencing and contig assembly have recently been developed 9,10 . These technologies still rely on random DNA shearing but enable the separation of individual template molecules on beads and their independent amplification in a cell-free system by emulsion PCR 11 . Sequencing is subsequently performed on each bead, using pyrosequencing 12 or sequencing by ligation 10 rather than the traditional Sanger method.

However, the use of such integrated sequencing pipelines seems inappropriate when applied to RNA virus genomics. First, viruses are small, intracellular parasites for which large amounts of the targeted genetic material must be separated from the host nucleic acids to construct the library. This requires preliminary steps of tissue culture and ultracentrifugation before shearing and cloning 13 . In addition, the required yields of nucleic acids are difficult to obtain when working on RNA viruses and/or low-replicating viruses. Such a strategy is time consuming, difficult in the case of clinical samples and hazardous with human pathogens. Second, viral RNA genomes are usually small and/or segmented (1–10 kbp molecules). Third, RNA virus genomes frequently consist of short conserved motifs interspersed with long regions displaying high levels of genetic heterogeneity. Therefore, a common sequencing strategy consists of amplifying and sequencing long PCR products obtained using primers targeting the most conserved regions. For these reasons, standard viral RNA genomics has relied on 1–5 kbp RT-PCR amplifications followed by primer-walking, which involves de novo synthesis of specific primers to extend sequencing to undetermined regions. Characterization of a 4 kbp sequence by this method requires at least six rounds of sequencing. Design and synthesis of new primers make this strategy expensive and time consuming.

Alternative procedures can be divided into three groups. The first consists of replacing Sanger sequencing biochemistry with cheaper and faster technologies, such as pyrosequencing or sequencing by ligation-hybridization. However, their short read length, limited to 100 bp, is poorly adapted to high-throughput sequencing of short genomes. The second alternative avoids de novo primer synthesis by making use of a premade oligonucleotide library. Primers may be: (i) 5–10mer oligonucleotides randomly synthesized that are used either alone 14,15,16 or in a modular combination 17,18,19,20,21,22 or (ii) oligonucleotides resulting from high-throughput synthesis 23,24 . To date, the size of the required oligonucleotide libraries limits the use of these methods. The indexer walking 25 method has recently been proposed: DNA is submitted to cycles of digestions followed by terminal ligation of oligonucleotide adaptors ('index'). One end of the cloned DNA is sequenced with M13 universal primer, and this first sequence is analyzed to find specific endonuclease restriction sites close to its 3′ end. DNA is digested with the selected enzyme before ligation of a specific double-stranded indexer, amplified by PCR and submitted to Sanger sequencing primed with the indexer. These steps are repeated and allow unidirectional progress into the unknown sequence. As the indexer library is considerably smaller than those previously proposed, indexer walking might be a cost-attractive method as it avoids systematic primer synthesis. However, the procedure remains time consuming for the following reasons: (i) the choice of the indexer is a function of the nucleotide sequence determined in the previous cycle and (ii) experimental handling for a single cycle of digestion–ligation involves DNA amplification and DNA purification by gel purification, ethanol precipitation and streptavidin-coated beads.

Overview of the LoPPS technique

The third alternative uses the shotgun strategy to create, from a PCR product, a DNA library of fragments short enough to allow one-step complete sequencing. One major advantage of the shotgun strategy is the potential for automation of the entire process. Automation of bacterial growth, plasmid DNA purification and sequencing enables rapid sequencing of numerous large DNA genomes to high-quality standards with attractive cost profiles. We have developed the long PCR product sequencing (LoPPS) procedure (see Fig. 1) on the basis of this shotgun strategy. Three to five kilobasepair PCR products, generated by any standard PCR or RT-PCR method, are randomly sheared by ultrasound into ∼ 700 bp DNA fragments. The protruding termini are repaired to create blunt ends and 3′ A-overhangs added to allow TA cloning and to avoid fragment concatenation. The number of clones required for contig reconstruction is predicted from the length of the initial PCR product (see Table 1 and ref. 36). Clones are randomly selected and sequenced with the T7 PROM vector primer. Finally, the target sequence is reconstructed from overlapping fragments.


Supporting information

S1 Fig. Preprocessing of SCAN-seq data and quality control.

(A) Schematic of SCAN-seq data pretreatments (for details, see Methods). (B) The length distribution of cDNA products before library construction. (C, D) Length distribution of full-length reads. The numerical data are listed in S2 Data. SCAN-seq, single cell amplification and sequencing of full-length RNAs by Nanopore platform.

S2 Fig. Data quality of mESCs using SCAN-seq.

(A) Saturation curve of detected genes and isoforms in mESC. (B) Heatmap of correlation value (Pearson) between each pair of mESCs. The correlation value of mESCs by SCAN-seq was even better than that by SUPeR-seq and Tang 2009 method. (C) Coverage of reads along the whole transcripts. (A–C) The numerical data are listed in S2 Data. mESC, mouse embryonic stem cell SCAN-seq, single cell amplification and sequencing of full-length RNAs by Nanopore platform SUPeR-seq, single-cell universal poly(A)-independent RNA sequencing.

S3 Fig. Saturation curve of detected genes and isoforms in cells at different developmental stages.

The numerical data are listed in S2 Data.

S4 Fig. Gene expression analysis in mouse embryonic samples.

(A) Number of detected genes in each individual cell at each developmental stage/type. The numerical data are listed in S2 Data. (B) Correspondence of stage-specific genes detected using SCAN-seq and SUPeR-seq. (C) GO analysis of the 6 group of genes in Fig 3D. GO, gene ontology SCAN-seq, single cell amplification and sequencing of full-length RNAs by Nanopore platform SUPeR-seq, single-cell universal poly(A)-independent RNA sequencing.

S5 Fig. LncRNAs detected in mouse preimplantation embryos.

(A) The reads ratio of mESCs and all blastomeres at different developmental stages. The center represents the mean, and the error bars represent the SEM. (B) Heatmap showing the expression levels of LncRNAs in all cells. (A, B) The numerical data are listed in S2 Data. lncRNA, long noncoding RNA mESC, mouse embryonic stem cell SEM, standard error of the mean.