#HOTnet | Explore Tumblr Posts and Blogs

bloodorangesoup · 3 years

Text

Ice Cream and Bad TV | B.B.

Request: Late night thoughts : The only porn bucky watches are the ones that come on tv at like 2 or 3 in the mornings. Imagine cuddling up with him on his living room floor, aimlessly clicking around until you stumble upon the channel & he gets flustered & shy about it & you fuck him while porn plays in the background. Like I think it’s so romantic🥺🥺 riding him while the soft glow of the tv illuminates his face. His moans & grunting blending in with the actors 😫😫

Pairing: Bucky Barnes x Reader

Word Count: 3.6k (this was supposed to be a drabble but I cannot for the life of me control myself)

Warnings: NSFW 18+ | cuddle fucking, unprotected sex(pretend ur on birth control for a sec)(still wrap it b4 your tap it), flustered Bucky, porn talk, the tiniest bit of sub!Bucky for like half a second, fluffy smut, you get the gist

My Masterlist

Notes: I did this in one sitting so sorry if it’s repetitive or has any mistakes. I think I have a major soft sport for flustered Bucky. I also had to think of a realistic name for a TV porn channel so bear with me. This is my first time writing smut in non-headcanon form so let me know how I did! Happy reading!

It was 2am on a Thursday night when your phone rang with your boyfriend's caller I.D. This wasn't the first time that Bucky had called you for this instance, it became a routine for you to talk him down after a nightmare and you prepared to do so when you picked up. Usually the first thing you would hear was his heavy breaths, the anxious buzz in his body making him fumble his words as he tried to focus on talking to you, so you were surprised to be met with still breathing and his typical deep, steady voice.

"Doll? You there?"

"Yeah, Buck, I'm here," you replied with a yawn, trying to sound as awake as possible. Bucky could hear the airiness of your voice and let out a sigh.

"Sorry, baby, I woke you up."

"You say that every time, I don't mind waking up for you. Besides, I don't have work tomorrow so I don't exactly need my beauty rest."

There was a pause. You could still hear static from the other side of the call but Bucky didn't say a word.

"Buck? You okay?" As if you snapped him out of a daydream, his hurried words rushed through the phone.

"Yeah yeah, sorry. I'm kind of out of it right now, I had another bad dream."

"Do you wanna talk about it?" You had expected his usual answer of yes, then you would listen to his thoughts and comfort him through the horrific reality that were his nightmares.

"You said you don't have work tomorrow? Do you think you could come over right now? Actually never mind, I don't know what I'm saying, it's 2am." You could practically see Bucky shaking his head as he took back his question.

"I'll be there in twenty, James. You better have some blankets and pillows ready for me because we're taking over your couch."

Bucky let out a breath over the line, "See you soon, y/n, love you."

"Love you more," you said quickly before hanging up, not letting him get in the last 'I love you.'

Forty minutes later you found yourself on Bucky's lap on his living room floor. You two had abandoned the idea of the couch and chose to make a small fort with the couch as your scaffolding. There was a surprising amount of pillows and blankets surrounding the two of you, you had no idea Bucky even owned them all.

He sat with his back against the couch, your legs laid over his and your arms were around his neck, bringing his head down to your chest. He talked about his nightmare, sparing you the gruesome details, and once he was done he closed his eyes and leaned further into your chest, breathing in your scent. You gave the crown of his head a kiss and rested your head atop his. Comfortable silence washed over the two of you before you spoke up.

“You know what always makes me feel better?” you asked, lifting your head just enough to look down at him.

“What?” he mumbled into your chest. A soft smile graced your face at his cuteness.

“Ice cream and bad TV!” you announced excitedly before wiggling out of his grasp.

You did a little jog to his kitchen and reached down into the freezer to grab a small pint of ice cream you knew Bucky always kept stashed for you whenever you came around. Picking up two spoons and turning off all the lights in the apartment on your way, you quickly made your way back into the living room, lifting Bucky’s arms to situate yourself back into your position on his lap. You popped the lid of the small tub and handed it to Bucky along with a spoon. With your hands now free, you felt around in the dark for the remote and turned on the TV.

Waiting for the TV to completely turn on, you ate a spoonful of ice cream. You hummed at the taste of the cold sweetness coating your tongue. You gasped as you opened your eyes, finding Bucky scooping out some ice cream and lowering it to your mouth.

“Oh wait, let me make this cuter!” you squealed with your eyes wide.

Copying Bucky’s actions, you took a spoonful and lifted it to his lips, the both of you opening wide and feeding each other. You giggled at the cliché moment and Bucky couldn’t help but let out a chuckle at how adorable you were.

You turned back to the TV, lifting the remote in your hands to change the channel to anything but the news that was already playing on the screen. As you flipped through sports recaps, music channels, infomercials, and movies that were already almost over, you sighed.

“Ugh, there’s nothing good on. Maybe if I start from the end of the list there’ll be better stuff,” you huffed.

Before Bucky could register what you said you had already typed in the biggest number you could enter and began flipping down through channels. Surfing through the empty screens, you landed on one called HotNet and suddenly the screen was completely taken over by a pair of boobs which then cut to a woman on her back and a man on top of her, rutting into her at a slow pace, while her moans echoed throughout the apartment. You quickly shuffled with the remote, trying to turn the volume down from it’s loud setting, spooked at the loud, obscene noise coming from the speaker.

“Oh my gosh, I wasn’t expecting that,” you laughed into Bucky’s chest, “God, I hope your neighbors didn’t hear that.”

Bucky stared at the screen with wide eyes, hoping you would change the channel before noticing that the channel was in the On Demand section. You gasped through your laugh, collecting yourself before turning back to the TV and shaking your head.

“Jeez, I didn’t even know they still had porn like this on public television.” You looked back down at the remote, clicking the Guide button. Bucky’s heart stopped as the description took over the screen, the video still playing in the top right corner. In bold letters the words On Demand titled the card. Even worse, the bottom of the screen displayed a small box containing the information,

$19.99

purchased with debit card xxxx-9758

You furrowed your brows in confusion, realization slowly softening them, before a mischievous smirk took over your face. You lifted your head to look at Bucky, his face looked down at your lap, refusing to meet your eyes. You tapped the bottom of his chin with the remote, lifting his head to meet your gaze.

“Did you order TV porn, Jamie?” He could hear the teasing tone in your voice, and you only ever used that nickname when joking around, yet Bucky still felt extremely embarrassed. He felt as if he just got walked in on while touching himself. It didn’t help that it was you that saw it, he wanted to crawl in a hole and never look back.

“Ah, you’re blushing,” you squealed as you took his cheeks in your hands, “oh come on, it’s not that embarrassing Bucky.”

“Ugh, can we please pretend you didn’t just see that,” Bucky groaned, closing his eyes to escape your stare. He set down the ice cream next to him and lifted his forearm over his eyes.

You let out a breathy laugh, grabbing his shoulders to stabilize yourself and pushing up to swing your leg under you to the other side of his lap, you straddled him.

“Hey, it’s really not that bad. I mean we’ve had sex before Buck, I think you’d rather have me find this than Sam or someone else.” He let out another groan and lowered his head to lean against your neck.

“Angel, please don’t talk about Sam while you’re on top of me and there’s porn playing on the TV.”

You laughed and ran your hands down his arms, letting one of your hands run up behind his neck and weaving your fingers into his hair. You gripped the back of his head and gently tugged him back up to face you.

“Why,” you sang, deciding to have some fun with him in his flustered state, “is this turning you on?” you whispered the last part.

Before he could respond you leaned down and kissed him. You didn’t bother to start off slow as you usually would, Bucky could taste the hunger from every parting of your lips. His hands gripped your waist, you rocked your weight forwards to rub against him. Bucky’s head clouded, his senses felt overloaded in the best way possible, your movements mixed with the sounds of the moaning coming from the TV were getting him hot. You pulled back before getting too carried away, a smile on your swollen lips.

“So, baby, tell me about it.” You said. It was Bucky’s turn to furrow his brows and he cocked his head to the side in question, mind still foggy from the feeling of you grinding on him.

“What kind of porn do you watch?” you clarified, looking at him like you just asked what his favorite color was.

“Uhm, you want to know about that stuff?” Bucky looked at you with caution, feeling out if he should listen to you or not.

“Well, yeah. I mean I don’t know how it was back then, but most people nowadays watch porn. It’s not super taboo to talk about it,” you explained. You weren’t going to force him to talk about it if he really didn’t want to, but your desire to hear what Bucky was into was strong.

He hesitated for a moment, looking in your eyes and seeing curiosity clouded with lust. He cleared his throat.

“Well, pretty much this stuff,” he gestured with his hand to the TV before putting it back on your waist, “there’s this girl on there, she kinda looks like you, so I buy the videos with her.” He closed his eyes and internally groaned at his words, he didn’t mean to come off so perverted. God, you had already found his porn, the last thing he needed was for you to be thinking he was a creep.

“Y’know that’s actually kinda sweet,” you giggled, “what do you like about those videos?”

Feeling a bit more comfortable knowing you weren’t grossed out, Bucky continued.

“I like the ones where it’s dark and slow. It’s usually quiet and there’s candles and stuff,” he explained shyly. Of all the surprises that came with dating Bucky, you had to admit finding out he was into romantic porn was one of the biggest ones.

Bucky cleared his throat again before questioning you, “Do you watch porn?” He felt almost wrong asking that. He knew that women in this age were more sexually liberated, but the words still felt sticky in his mouth.

“Oh, yeah, of course I do.” Bucky seemed surprised at how casually you answered. He raised his eyebrows, silently asking you to go on.

“Well specifically, I actually really like the videos that are like this,” you said, your head looking down at you straddling him, he followed your gaze, taking in how nice you felt on his lap. You leaned forward, bracing your hands on his chest. Your faces were so close your noses were almost touching. You could feel his rapid heart beat under your palm and the quick rise and fall of his chest. “Do you ever watch the ones that look like this, Bucky?” He looked up at you and nodded with wide eyes, his pupils were blown and his mouth was open like he was searching for the words to respond.

You grasped his jaw in your hand, your thumb on his chin, and looked him in the eyes. You wanted nothing more than to jump his bones, but with the newfound knowledge you had, you took your time with your actions. Within the last five minutes, Bucky had revealed to you not only that he liked soft, romantic porn, but that he also watched a specific actress because she looks like you. The one goal in your head was to fulfill his fantasy the best you could at the moment.

With his head still in your hand, you held your eye contact as you let your knees move out, grinding yourself against his growing erection. He let out an involuntary whimper. That was the last straw. You pulled his face to yours, joining him in a hard, yet loving kiss. Your arms wrapped around his neck, pushing your chest against his, trying to get as close as possible.

Bucky’s dick was painfully hard. He couldn’t focus on anything but the feeling of you on top of him. His body felt like it was on fire, he was insatiably horny and couldn’t control the way his hips bucked up to grind with you. He felt like a horny teenager the way his mind was filled only with images of you naked, imagining you like that on top of him like you were now.

You slipped your tongue past his lips, keeping your pace slow as you glided it across and bit at his lower lip. A moan made its way out of your mouth and into his as you felt him buck up against you.

You slid your hands down his chest, fumbling with the hem of his shirt before his arms raised, giving you access to peel it off him, throwing it up on the couch. Bucky kissed down your neck, licking over the shell of your ear, and sucking at the center of your throat. He reached down and slipped his hands under your shirt, sliding up and down your waist before gliding up and lifting your shirt over your head, discarding it with his.

Bucky wasted no time reaching around you and unclipping your bra and dragging it down your shoulders. He cupped your breasts, squeezing them with his large hands before bringing you into another tender kiss. Despite your frantic movements, there was a spark of passion cracking through the air, the both of you desperate to feel each other closer.

You broke the kiss, breathing heavily and resting your forehead against his.

“I wanna make love to you, y/n, please, let me make love to you,” he groaned with a slight whine in his voice. He sounded like he was about to crack.

“Please, Bucky.”

You leaned back on your hands and lifted your hips, giving Bucky the room to pull down your shorts and panties in a few swift tugs. He quickly got up on his knees and pulled down his sweats and boxers, sitting back down and tugging them off his legs. You reached your arms forward, Bucky grabbing a hold of your wrists and pulling you back to straddle him again. You gasped at the feeling of his cock under your bare pussy, your wetness from all the grinding and kissing making you slide over him.

You leaned forwards, capturing his lips between yours and raising yourself up on your knees. Reaching under yourself, you gripped his cock, wet with your arousal, and lined him up with your cunt. Bucky gripped your hips as you sank down on him, using every ounce of self control to stop himself from rutting up into you. You stayed like that for a second, adjusting to the way he filled you. Seeing the remote lying on the floor, you grabbed it and slightly turned up the volume, only enough so that you and Bucky could hear the actress in the video being ravished by her co-star.

Bracing your hands on his shoulders, you began to slowly move up and down over him, riding him just as you had told him you liked to watch. His hands moved up from your waist, squeezing your tits, pinching your nipples in between his fingers. You let out a hiss as he rolled them between his fore finger and his thumb, tugging on and rubbing his thumbs over them. He reached around to grab your ass, squeezing and rubbing your cheeks and you used him to pleasure yourself.

You were a moaning mess over him and he wasn’t much different. The room was filled with the sounds of the porn playing on the TV mixed with the sounds of your own cries of pleasure. If the neighbors hadn’t been woken up by the TV, they sure were by you two.

Bucky sat back on his knees, his hands slid down to your thighs, moving your legs from under them and wrapping your legs around his back. His arms worked their way back up to wrap around your waist, bringing you ever so close. The new angle pushed him even deeper up into you. He buried his head in the crook of your neck, giving you access to all the sweet, desperate sounds falling from his lips.

You were now pushing up with your feet behind him, rolling your hips over his. The new technique made your clit rub over his pelvis, the sensation making you throw your head back and let out a deep moan. He took the opportunity to continue kissing your neck, going lower with every lick and bite. He sucked at your collarbone, leaving a deep purple mark in its place.

Suddenly, he was using his arms to lift you up, not pulling out of you, and laying you down on the blanket-covered floor. You instinctively locked your ankles together, pulling him closer as he thrusted into you. Bucky had his metal arm holding himself up while his flesh one squeezed your breasts, feeling them move with his thrusts. He lowered his head to your chest and licked one of your nipples, grazing his teeth over it before taking it in between his lips and sucking. Your hands flew to the back of his head, singing praises as you wove your fingers into his hair. He continued to alternate between your breasts, leaving love bites around them and sucking on their sensitive peaks.

You could feel your body buzz with anticipation, you were so close. The moans escaping your mouth were lewd and uncontrolled. Bucky wrapped his flesh hand around your waist, lifting your chest flush against his.

You snaked your arms around his neck, pulling yourself up to kiss him. Nothing felt better than making love with Bucky felt. This was definitely not the first time you two had been intimate, but none of those previous times had ever been like this. You could feel the passion flowing through his body into yours. You looked up at his form over you, how beautiful he looked with the light of the TV screen glistening against the beads of sweat on his forehead. There was absolutely nothing in the world compared to having him, in all his beauty, to yourself like this.

Bucky’s heart was exploding in his chest. What had started out as an embarrassing, nightmare-fueled night had turned into the most intimate experience he ever had in his life. Love was already established in your relationship, but Bucky had never truly made love to you. He looked down in awe at your disheveled state, at the way your hair was sticking to your forehead, at the way the only light in the room gave you an angelic glow. He wanted nothing more than to give himself completely to you, to make you come all over him.

He leaned his forehead against yours, pushing his face forward once every few seconds to kiss you. He was close, and he wanted you to come with him. He leaned into you even more, his cheek pressed against yours, his lips next to your ear.

“I love you so much, y/n, God, you feel so good. I love you, I love you,” Bucky’s voice was deep and strained, his words being emphasized by each push into your wet pussy. You let out another loud moan.

“I love you, James,” you whined as he hit your sweet spot, “I love you so much.”

Bucky’s hand wedged itself in between the two of you, his fingers finding your clit and gently rubbing it. You were completely overwhelmed, this is what it felt like to make love to someone. Your body began to shake and your breathing became erratic. Bucky thrusted harder, working to push you over the edge.

“I want you to come in me, Bucky,” you gasped as his thrusts got faster and lost their rhythm.

Your mouth turned open in a silent scream as you came. Bucky felt your walls clench around him, squeezing the life out of him. He let out a shaky groan as he fell over the edge. His eyes shut tight and his body tensed as he spilled into you.

Wrapping his metal arm around your back and securing your body to his, he rolled on his flesh shoulder onto his back. You went limp against him, laying your head on his chest, listening to his heart beat slow down. He was still buried within you, connected to you. Letting your eyelids fall shut, you savored the feeling of being full of him and his love.

You lifted your head to kiss his jaw.

“I’m glad you called me, Bucky. I love you.”

“I am too, doll. I love you more.”

#bucky barnes smut #bucky barnes fluff #bucky barnes x reader #bucky barnes imagine #bucky barnes fanfiction #bucky barnes x y/n #bucky barnes x you #bucky barnes x f!reader #bucky barnes x female reader #bucky barnes fluffy smut #bucky barnes one shot #bucky barnes

665 notes · View notes

fiamh · 5 years

Text

SESSION 7: BIOINFORMATIC ANALYSIS OF CANCER GENOMES

The final session of the meeting, chaired by Nuria Lopez-Bigas, starts with the Bioinformatics Challenge results from Mike Schatz.

Pan-cancer network analysis of combinations of somatic mutations

Ben Raphael (Brown University) follows Mike with a slightly revised talk on mutational heterogeneity. Recap of driver mutations, intra-tumor heterogeneity, long tail discussion and inter-tumor heterogeneity. Announces a “whirlwind tour” of methods developed in his lab (uhoh).

Can we infer tumor composition from single, mixed tumor sample? Number of SNV- and CNA based methods (shows dozen of references from ABSOLUTE to TITAN to PyClone and more, 2014 the year where this exploded). Copy number aberations used to be based on SNP arrays, shift to NGS. Detect subtle shift of red depth, combine signals across multiple aberrations and subpopulations (probabilistic algorithm THetA, recommends reading the paper for optimization tricks, works for ~7x WGS data). Example from ~200x BRC samples with 3 subpopulatins and how THetA automatically deconvolutes them; another example from whome-exome data (compares favorably to ABSOLUTE).

Wanted to go further to study tumor evolution process using variant allele frequencies. Noisy for single mutations at lower coverage, but can be clustered across genome (dirichlet process mixture models) and used to create the original tree. Can’t measure leaves without single cell data, but can convert allele frequency histogram to create the evolutionary tree using binary tree partitions (assumption: only one clonal expansion event at a given time). Simulated data examples look reasonable (but only around 50% precision at 8+ populations).

Next compare mutations across different genomes. Ranked list of significantly mutated genes — how to handle the long tail? Genes don’t act on their own, univariate analysis not ideal. Analyze gene groups instead and use topology of gene interactions: aim to discover pathways given enough samples, but can’t do it without any prior information. HotNet and Dendrix work on that intersection to find significantly mutated subnetworks. Consider gene score and interaction scores at the same time (HotNet, HotNet2 using heat diffusion methods to spread significance scores across network, with HotNet2 using directionality information). Applied to TCGA pan-cancer project to find consensus subnetworks, e.g., cohesin complex, condensin complex, SWI/SNF identified. Mutual exclusively another story they have been working with. What about mutually exlusive gene sets (Dendrix and now multi-dendrix for multiple pathways, and Dendrix++ with probabilistic scoring).

Also started looking into some visualizing approaches with d3.js just out of necessity. Overall aim is to combine different data types, get better at stratifying samples.

Ginkgo: Visual analytics for single cell CNV analysis

Somehow I missed the announcement of Tyler Garvin’s talk (from Mike Schatz’ lab) . Quick summary of single cell sequencing, CNVs in cancer. Underlying concept are CNV profiles for each sample/cell, allowing you to cluster patients or individual tumors and explore the resulting tree. Data is quite noisy due to whole genome amplification, wet lab protocols, sequencing biases. Correct for outliers, provides lots of QC metrics for each sample/cell, clustered and visualized. [Followed by demo of the ginkgo software using data from Navin et al (2011). Framework looks good, but again hard to live-blog.]

Validated with five public data sets, easily reproduced results in ~30 minutes. Compares GC biases across different data sets and data dispersion, and the need to correct for these. Couple of recommendations: use DOP-PCR for WGA, sequence healthy diplied cells, plan 25% cells to be filtered out and plan accordingly, save your FACS data (get estimate of ploidy from staining), start with 500kb bins, 100 reads/bin (1.5 million reads per cell) and do control for gender.

Mapping intra-tumor heterogeneity using multidimensional single cell data

I’m scared of trying to cover [Dana Pe’er]’s talk (Columbia University) given the amount of information she tends to pack into a single presentation. Talk on work with the Silva lab (Felix Sanchez-Garcia). Unclear what went wrong for a primary tumour (dissecting the plane crash: what went wrong, what do the broken parts do), vs functional approach (analyze individual features). Integrative approach (Helios) to combine the best of both approaches. Gives examples of oncogene addiction, Helios scores neighbouring genes to come up with best candidates. Training sets too small and biased, using an unsupervised model with frequency of alterations as key. Quick detour to copy number modeling and how to integrate these and other features (EM feature learning to identify important features and associated drivers).

Shows how it pinpoints BCL2, 6th based on CNA but oncogene addiction raises it to top of peak. Looked at 17 most frequently amplified regions and test them one by one. In vitro validations tends to score known candidate genes highest, cloned picked genes for genes without known targets in six replicates to see if they drive tumor development. 10/12 genes validated, overall 93% accuracy.

Switch themes to intra-tumor heterogeneity. Mass cytometry as a game changer looking at millions of single cells, label biomarkers with metal isotypes, 45 dimensions simultanously measured in millions of individual cells (see viSNE paper for details on data analysis of healthy immune system, detecting rare cell populations in ALL and more). Now to the unpublished stuff (with Nolan lab at Stanford). Ceullar profiling of an AML cohort vs healthy bone marrow. 28 samples, 19 perturbations, 16 million single cells. Must clustering approaches fail for this kind of data. Don’t know number of clusters, size assumption, unclear distribution. Graph-based clustering to tackle the toplogy, each cell a node connected to most similar cells (similar to already published work). Their ‘PhenoGraph’ seems to be doing better than competing methods (tested on healthy data). AML graph landscape is mixed between patients. Each patient resides in multiple areas of the map, each region contains multiple patients. Some structure to the aberrant surface expression of AML, visible when color-labelled by surface marker expression: developmental gradient apparent.

Try to find ‘metaclusters’ shared across patients, iterate on graph to find clusters of clusters. Each patient has its own combination of (repeated) metaclusters. Samples with similar genetics have similar meta-cluster profiles, i.e., genetics is driving some of the big structure but not the difference between the subpopulations. These seem to map to intracellular signaling structure — response to drugs, growth factors and other perturbations. Matrix of 16,000,000 x 31 cells reduced to tiny fraction to learn surface-signalling axis is disrupted in AML, some CD34- populations signal like stem cells (surface, signalling link uncoupled). Signalling data can be used to classify healthy cells successfully. Try to find AML subpopulations signalling like healthy cells [and I got lost at this point.] Possibly new definition of ‘primitive’ cell, % greatly differs between samples and does not agree with CD34+ fraction currently used to describe them. Map to gene signature, then used gene signature to test in much larger data set, linked to survival.

[I need a break now…]

Structural variation analysis of tumor genomes

Peter Park (Harvard Medical School) with the last ‘full’ talk of the session (another disclaimer: like Nils Peter is a collaborator). Quick summary of SV events in cancer (refers to Nils who made some of the graphics of different events). First part of the talk on published work regarding complex SV events, e.g., duplication with deletion. Need to look at all events combined to understand the actual event. Tons of manual curation to find unusual / novel cases. Found SV in TERT promoters (part of the TCGA work), asked whether they can infer mechanism based on sequence homoogy at breakpoints (fork stalling, template switching, non-homologous end-joining, etc., see work from Kidd et all, Cell 2010 for HapMap). Try to do this in short-read cancer data. Nice summary of somatic SV types and mechanism spectrum (by frequency) from 140 samples.

Characterized GBM, reconstructed complex re-arrangements in detail. About 20% of somatic deletions are complex formed by replication errors. Many focal deletions caused by FoSTeS/MMBIR, both DNA DS-breaks and replication erros drive somatic complex events. Amazing reconstruction graph involving 45 events that might explain a single EGFR event, trying to proof that this is the only way to get there, but no automated way to generate these yet.

Switch to transposable element analysis pipeline (see paper), checking for somatic mosaicism in brain based on data from Chris Walsh. Is there somatic variation in different neurons, is there retrotransposition? Genome mosaicism driven by retrotransposition might reshape genetic circuity. Whole genome amplification, sequenced L1-sequencing of 300 single neurons to detect L1 insertion sites. Observed ~.6 insertions / cell, much lower than previous publications. Sequenced 16 cells at 40x (MDA) compared to populatin of 100 neurons and bulk data. Compares MDA vs MALBAC, biases. Retrotransposon detection workflow in the paper, show L1 insertion in same places across muliple neurons (expect to be the case if they have the same progenitor, validated). Think close to 80% sensitivity in single cell compared to bulk. Found additional insertion missed by L1-seq (longer than DNA-fragments captured by L1). See another recent paper on impact in cancer genomes.

Other bits and pieces: nozzle shown, used by the Broad for Firehose (and other NGS reports). Shoutout to Refinery, a repository framework on top of Galaxy (work with Shannan Ho Sui in our group). Facetted browsing for modENCODE data. Followed by list of ~10 other tools (Meerkat, BIC-Seq, StratomeX again, etc.).

Using de novo assembly to exhaustively catalog tumor mutations

A selected talk from David Jaffe (Broad Institute) on mutations only seen by de novo assembly. Start with cheap data, 0,5ug of DNA, PCR-free library, paired end read 50x, 250bp from breast cancer cell line, but also tested on primary tissue. DISCOVAR de novo is the algorithm, tumor and normal assembled together, mutation caller still work in progress and unpublished. Walks through the graph assembly algorithm. Bubbles represent alternative paths including somatic mutations. Look for tumor only edges, reads from just from the tumor not the normal sample. Compare against MuTect, Strelka, Taiga. [Live demo having minor difficulties due to having to reboot his Mac and of course getting logged out of all his sessions]. DISCOVAR finds ‘funky’ nversions not found by standard methods — reads can’t be aligned. Also found a ‘crazy quilt’ rearrangement (love the names). Re-arrangement supported by 140 reads in tumor, trace in assembly which goes through 13 segments all across the genome, each 100-200 bp.

A general mutation finder that sees invisible somatic mutations, more than 500 of these found in HCC1143. Want to test drive on interesting samples, volunteers welcome.

I did have to miss the last talk of the meeting from Hossein Khiabanian on Moduli spaces of phylogenetic trees describing tumor evolutionary patterns unfortunately.

Thanks to the organizers and chairs for a terrific meeting. Just the right size for networking and a good mix of talks!

0 notes