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By Pooja Toshniwal Paharia Sep 22 2023 Reviewed by Danielle Ellis, B.Sc.

In a recent study published in Nature Communications, researchers proposed a simplified approach for analyzing messenger ribonucleic acid (mRNA) vaccines using long-read sequencing. Study: mRNA vaccine quality analysis using RNA sequencing. Image Credit: Jo Panuwat D/Shutterstock.com Background

Messenger RNA vaccines demonstrated safety and efficacy during the COVID-19 pandemic, but extensive quality and purity testing is required to verify their efficacy and safety. Manufacturing advances have enabled billions of doses to be manufactured with acceptable quality and safety.

Various approaches are now utilized to assess mRNA vaccines; however, the efficacy of novel therapies depends on speedy and safe manufacture. Rigorous analytics are required at each stage of the production process to detect impurities and assure the safety of mRNA vaccines. About the study

In the present study, researchers investigated the VAX-seq method for quality analysis of messenger RNA vaccines.

The researchers developed VAX-seq, a simplified procedure for analyzing mRNA vaccines and therapeutics using long-read sequencing. This procedure compares VAX-seq to industry standards, including chromatography, capillary and agarose electrophoresis, and immunoblotting. The researchers employed a variety of methodologies, including Illumina plasmid DNA sequencing, ONT cDNA-PCR sequencing, and Oxford Nanopore direct ribonucleic acid sequencing.

Key messenger RNA quality features assessed by VAX-seq were sequence similarity, integrity, 3'-poly(A) nucleotide tail dimension, and RNA and DNA contamination. To assist VAX-seq, a software toolbox was created that provides thorough and automated reporting on mRNA quality. An enhanced green fluorescent protein (eGFP) messenger RNA was created and generated as a reference to demonstrate the application and validity of the methodology,

The plasmid template was amplified in Escherichia coli, isolated, purified, and linearized as the initial stage in the preparation process. The linearized pDNA template was then employed as a template for synthetic mRNA transcription in vitro. To examine the isolated mRNA, the program was combined with complementary deoxyribonucleic acid (cDNA) sequencing. VAX-seq attached a reverse transcriptase primer to the 3' terminus of the poly(A) nucleotide tail, allowing the length of the tail to be measured.

The researchers used the tailfindr program to normalize deletion mistakes and the read-specific nucleotide translocation rate. As part of the VAX-seq process, the complementary DNA library preparation introduced two flanking-type adaptors to the messenger RNA's 5' and 3' ends.

To identify complete-length molecules of mRNA from truncated messenger RNA, sequencing reads contained both flanking adaptors. Off-target RNA contaminants were identified using VAX-seq, which was used to assess fragmented and off-target RNA contaminants in cDNA libraries. Results

The analysis revealed that VAX-seq, a technique for sequencing mRNA vaccines, can detect sequence, length, integrity, and purity. It also enabled the examination of linearized plasmid DNA templates and the detection of impurities from plasmid amplification. VAX-seq easily established the length and similarity of mRNA vaccine sequences. The eGFP mRNA size profile revealed a major peak (77%) that was within 5.0% of the predicted length [1,153 nucleotide (nt)-long], as well as a varied spectrum of smaller, fragmented mRNAs. Short-read sequencing provided insufficient and inconsistent coverage, while heterogeneous alignment coverage was highly repeatable across replicates. Related StoriesDoes IQ influence COVID-19 vaccination decision-making?Inactivated poliovirus vaccine elicits persistent immunity for up to 10 yearsModernas adapted COVID-19 vaccine that targets Omicron XBB.1.5 approved by MHRA

Most sequences were aligned with the on-target messenger RNA product, and only a few reads revealed Escherichia coli contamination. The remaining seven percent of ribonucleic acid species were off-target RNA molecules, with 0.3% presumably originating from initiation sites of cryptic transcription. Direct ribonucleic acid sequencing libraries produce lower yields than comparable complementary DNA sequencing genetic libraries and cannot be multiplexed at the moment.

The researchers did, however, identify biases particular to direct ribonucleic acid sequencing, such as inferior-quality poly(A) nucleotide tail deletion. Direct ribonucleic acid sequencing found changed nucleosides in messenger RNA vaccines, demonstrating that including modified nucleosides in mRNA vaccines might lessen the innate immunological response while improving stability and translation.

Modified nucleosides had minimal effect on messenger RNA quality features and complementary DNA sequencing errors between messenger RNAs, including native N1-methylpseudouridine and uridine, but direct ribonucleic acid sequencing had a larger error rate.

Complementary DNA and direct ribonucleic acid sequencing revealed that modified messenger RNA vaccines had more truncated-type transcripts, with 41% complete-length and 54% truncated messenger RNA molecules, especially those less than 500 nt in length. Direct ribonucleic acid sequencing discovered nucleosides of N1-methylpseudouridine with a distinctive base-calling mistake that miscategorized N1-methylpseudouridine into cytosines, skewing the messenger RNA length profile. Conclusions

Overall, the study findings showed that VAX-seq was a procedure based on sequencing long reads that assessed essential mRNA quality characteristics such as integrity, contamination, and sequence identity. This technique can potentially become important to developing and producing mRNA medicines, offering a thorough and integrated evaluation at various manufacturing stages. VAX-seq employed full-length complementary DNA sequencing using Nanopore chemistry, which allowed for accurate assessment of the poly(A) molecular tail length as well as various off-target readings.

The approach offered a sensitive and quantitative assessment of mRNA characteristics, making it a more efficient alternative to conventional analytical techniques. VAX-seq enabled real-time identification of antisense RNA and messenger RNA integrity, allowing for swift testing lasting a few hours post-manufacture.

It might also identify complicated off-target ribonucleic acid contaminants created during transcription in vitro, as well as the degradation or sharing of messenger RNA vaccines during manufacturing, storage, and transportation. VAX-seq needed only a small quantity of messenger RNA as input and may be integrated to allow for large-scale and low-cost validation of vaccination batches. Journal reference: Gunter, H.M., Idrisoglu, S., Singh, S. et al. mRNA vaccine quality analysis using RNA sequencing. Nat Commun 14, 5663 (2023). doi: https://doi.org/10.1038/s41467-023-41354-y https://www.nature.com/articles/s41467-023-41354-y

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Brewers celebrate division title, feel like underdogs

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Brewers celebrate division title, feel like underdogs

ST. LOUIS — As celebratory prosecco and beer poured down his face, Christian Yelich soaked in the satisfaction of the Milwaukee Brewers‘ third straight NL Central Division title.

“This is the why,” Yelich said Sunday after the Brewers clinched, courtesy of the Cubs1-0 loss in Cincinnati. “This is why everything’s harped on throughout the year. Why we pay attention to detail, why there’s tough love throughout the season, why you keep grinding and why you keep trying to get better is for these moments — the fact that you can celebrate with your teammates.”

Carrying the best record in baseball, the Brewers won their fourth division title in the past five seasons and yet feel like the underdogs as the playoffs loom.

“In baseball, any team could beat anybody, especially in short series. Obviously, we’re not going to be favored. Everyone’s going to kind of point to our question marks and how there’s other teams that have more experienced players and, on paper, super talented rosters,” Yelich said. “But I think that gives our team a freedom. Nobody thinks that you’re going to be able to do this. So go out there and just play.”

Milwaukee lost Willy Adames to free agency and traded away closer Devin Williams in the offseason, and it was unclear what contributions veterans Brandon Woodruff (shoulder surgery) and Yelich (back) would be able to provide.

Both were able to be key players, although Woodruff’s status for the postseason is in question after he went on the injured list Sunday with what the team described as a moderate lat strain.

“Are we the most talented? No. Are we going to go up against teams far more talented with four times the payroll? Sure, but it doesn’t bother this team,” manager Pat Murphy said.

Since 2018, the Brewers have lost in the wild-card round four times, in the division series once and in the NL Championship Series once. The franchise’s only pennant came in 1982.

“I heard somebody say we’ve got the regular season figured out, now we’ve got to figure out the playoffs. That’s one of the most absurd comments I’ve ever heard,” Murphy said, emphasizing his squad’s body of work.

After losing 5-1 to St. Louis, the Brewers had to wait less than a half hour before the Cubs’ game ended. The team put on navy blue “division champs” shirts, sprayed bottles of chilled prosecco and dumped cans of beer on each other’s heads.

A blue flag with late announcer Bob Uecker’s signature hung in the soaked visitors locker room.

Owner Mark Attanasio suggested there was some “Brewer magic or Uecker magic” in this team.

“It’s really just the culture. We show up every spring training with the goal of winning the division,” said Sal Frelick, in his third year with Milwaukee. “So, it’s been great. I’m fortunate to be able to do it every year.”

The Brewers gathered on the Busch Stadium infield for a team photo and were about to disperse when Murphy came up the stairs from the dugout, almost left out of the moment.

“When we started the year, nobody thought we’d be here at this point. Obviously, we’ve had our share of injuries. Nobody could have imagined we’d be in this spot right now,” Woodruff said.

The Brewers were at their best during a 29-4 stretch in July and August, turning a four-game deficit into a commanding nine-game advantage in the division.

“We’ve done a heck of a job as a group to get to this point,” Woodruff said. “And you know what? There’s still a lot of baseball left for the Brewers.”

Last year, they won the Central by 10 games before dropping the wild-card series to the Mets — losing the decisive Game 3 after Williams squandered a two-run lead in the ninth inning. Pete Alonso‘s three-run home run was the key.

To Murphy, who took over as Milwaukee’s skipper before this past season, that’s the fickle nature of the playoffs.

“It takes a lot of things to go your way, and sometimes having great talent helps that,” Murphy said. “I don’t worry about this team, and I don’t worry about what anybody says about this team. I get to live it every day and see how special they are.”

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First AL ticket punched as Jays earn playoff spot

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First AL ticket punched as Jays earn playoff spot

KANSAS CITY, Mo. — The Toronto Blue Jays became the first American League team to secure a spot in the postseason on Sunday with an 8-5 victory against the Kansas City Royals.

The AL-best and AL East-leading Blue Jays locked up a playoff spot with a week remaining in the regular season after a less-than-stellar start of 16-20 in early May and trailing by as many as eight games in the division in late May.

“I remember back when we were in Tampa in May, we weren’t playing very well and we got swept there,” Blue Jays manager John Schneider said. “I think these guys did a great job of rallying around each other, but the turning point was really when we came out of Tampa and went into the Texas series.”

This is Toronto’s third playoff berth in four years and fourth in six seasons. They missed the postseason in 2021 and 2024. Playoff success has been elusive for the Blue Jays, who haven’t won a postseason game since 2016. And, unlike the past three trips, they hope this year they won’t have to play in the AL wild-card round as they try to win their first division title since 2015 as they close out the regular season with a six-game homestand against Boston and Tampa Bay.

“You could feel it with this group in spring training,” Schneider said. “I know that sounds really cliché, but when you get a group of men that are committed to the same goal, you can do things like this.”

The Blue Jays’ 90-66 record is tops in the AL and they lead their division by 2½ games over the New York Yankees. If Toronto wins the AL East and has one of the two best records in the league, it will advance to the AL Divisional Series, which starts Oct. 4.

The last time Toronto made it that far was nine years ago.

“I’m just so happy for them,” Schneider said. “It’s hard at this level for everyone to put their egos aside and to play for one another. It’s so cool to see these guys completely happy for one another when they get the job done no matter who it is. This is the most fulfilling team I’ve ever been a part of with different characters, different skill sets, guys coming together for one common goal which is what’s important now. This is something you always celebrate.”

The Blue Jays are trying to win their first World Series since 1993.

“Today we go back to the postseason, but the journey is not over yet,” Vladimir Guerrero Jr. said. “We still want to win the division over the next six games. Since spring training, everyone has been together and when you see a team like that you start believing.”

Toronto snapped a four-game losing streak with Sunday’s win, and after the game popped champagne in the visitors clubhouse in Kansas City.

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Fading Mets slump into wild-card tie with Reds

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Fading Mets slump into wild-card tie with Reds

NEW YORK — For nearly six months, almost the duration of an entire Major League Baseball regular season, the New York Mets occupied a spot in the National League playoff picture. That changed Sunday after a brutal loss that concluded an ugly weekend and prolonged a baffling three-plus-month swoon with a week remaining on their schedule.

The Mets’ 3-2 loss to the last-place Washington Nationals at Citi Field — a sloppy, toothless showing cemented with two jaw-dropping catches by Nationals center fielder Jacob Young — combined with the Cincinnati Reds’ 1-0 win over the Chicago Cubs dropped them from postseason position for the first time since April 5 when their season was eight games old.

“It’s the way it’s gone,” Mets left fielder Brandon Nimmo said. “I can believe it because I’ve watched it. We’ve been watching it happen right in front of us.”

While both teams have an 80-76 record with six games remaining, the Reds hold the tiebreaker after winning the season series between the clubs. The Mets, who have lost 11 of their last 15 games, finish the regular season with a road trip against the Chicago Cubs and Miami Marlins starting Tuesday. Cincinnati hosts the Pittsburgh Pirates for three games before concluding their schedule on the road against the Milwaukee Brewers.

The Arizona Diamondbacks are also looming, just one game behind the Reds and Mets in the standings for the third NL wild card spot. Arizona owns the tiebreaker over both clubs.

“We just gotta win,” Mets first baseman Pete Alonso said. “It’s simple. Winning solves everything at this point. We just gotta do it. That’s it. We got to solve our issues between the lines. That’s the simple fact.”

The Mets, at 45-24, boasted the best record in the majors through June 12. They looked like a club that would sail into October after clawing their way into the playoffs and defying expectations with a trip to the NLCS in 2024 through 69 games.

But this year’s team has produced inverse results from the 2024 version that stumbled early before a magical summer pushed them deep into October. These Mets, with the second-most expensive roster in the majors, have gone 35-52 since June 13. That is tied for the fourth-worst record over that stretch with the Chicago White Sox. Only the Nationals, Minnesota Twins and Colorado Rockies have been worse over the 87-game span.

They enter their final two series with a 50.1% chance of reaching the postseason, according to FanGraphs. They were given a 96.2% chance on June 12.

“I can’t put my finger quite on it other than we just haven’t been able to put it together as a team for an extended period of time,” Nimmo said.

The Mets, coming off an encouraging series victory over the playoff-bound San Diego Padres, took the series opener Friday behind an offensive outburst. But they fell on Saturday in 11 innings on an inside-the-park home before before early mistakes, coupled with Young’s defense, buried them on Sunday.

In the first inning, Juan Soto, who went 1 for 2 with two walks, was picked off at first base. In the second, Francisco Lindor committed a throwing error that allowed the game’s first run to score.

“If we want to be where we want to be, those things can’t happen,” Lindor said. “Full accountability on that. I have to be better.”

And in the third inning, Cedric Mullins failed to take second base on a line drive that he hit down the left-field line, a decision that potentially cost the Mets a run.

Mullins said he thought the ball was caught by Lile as he crashed into the wall. Meanwhile, first base coach Antoan Richardson thought it was ruled foul. But the ball was ruled fair and bounced out of Lile’s glove.

Luis Torrens, who was on second base, was unsure if Lile made the catch so retreated to tag up and scored on the play as Lile writhed in pain. Mullins, despite teammates yelling and signaling to him to run to second base, remained at first base. He was doubled off moments later when Lindor cracked a line drive to first base. Soto then lined what would’ve been an RBI double.

“You just gotta go,” Mendoza said.

On the mound, the Mets deployed Sean Manaea and Clay Holmes as a piggyback for the second time in the last week after both veterans struggled to effectively pitch deep into games in the second half. The duo combined to give up just three runs — all in the second inning off Manaea — over six innings, with the biggest damage coming from the light-hitting Nasim Nunez swatting a two-run home run.

In the end, the Mets’ $38 million tandem was outpitched by the Nationals’ $1.4 million piggyback of Jake Irvin and Mitchell Parker, who entered Sunday with the highest ERAs in the majors among qualified pitchers this season.

They were buoyed by two highlight-reel catches from Young in center field. The first, a circus grab in which he kicked the ball to himself, robbed Brett Baty of extra bases in the fifth inning. The second took away a potential game-tying home run for Francisco Alvarez in the ninth.

“Those were crazy plays,” Mullins said. “In the stretch we’re in, every win matters. To see plays like that made, definitely deflates [you] a little bit. [We’ve taken] some tough losses on the chin. We have a week left. We’re doing to do some damage so that’s what we’re focused on.”

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