For our data snippet (again, high quality 30X coverage PCR-free 2x150 WGS), the effects of removing indel realignment are subtle. However, for data that deviate towards lower quality and for data that benefit greatly from BQSR, the effects could be more substantial. Given this, we ask researchers who are considering our new workflows to take into account properties of their data and consider the impact omitting indel realignment may have for their analyses. A first step towards this could be to examine the MAPQ distribution of the primary alignments.

I’m told these influences should be subtle for high quality data but make a difference for lower quality data

Any discernible patterns from misaligned reads around indels will be small compared to that of flowcell failures

wgs_hg19_125_cancer_blood_normal_panel.vcf. This PON was created with 125 whole genome samples derived using 2012 technology. The sample libraries were from the blood normal tissue of cancer patients. We do NOT use matched normal tissue samples, as matched normal tissue samples can be contaminated with tumor/pre-tumor tissue, as they are typically derived from tissue adjacent to the tumor. The samples are deep coverage samples, ~30x, aligned to hg19. The libraries were paired end and approximately ~101 bp reads (2x101).

you should at the least use samples prepared in the same manner (prep and tool-chain) as your samples and sequenced by the same center as your samples. The PON is meant to capture sequencing artifacts that may be different for different centers/sample-prep/tool-chains, and so matching its constituents closely to the provenance of your samples is ideal.

See this document for how to create your PON. Be sure to search our forum also if you have questions as there are multiple threads that help people with their PON creation.

We do NOT use matched normal tissue samples, as matched normal tissue samples can be contaminated with tumor/pre-tumor tissue

lowest MAPQ of reads indel realignment then increases is at MAPQ26 and in fact over 94% of increases are for MAPQ60 reads.

although they do not impact genotyping any more than uninformative reads, they may have a disproportionate impact on metrics that use exclusively informative reads

altered annotation metrics (QD, FS, SOR, MQ & MQRankSum for the snippet) impact cohort level annotation metrics, the variant quality score recalibration (VQSR) model and the resulting VQSLOD score for the site. These changes to the distribution of the variant scores impact variant site filtering

HaplotypeCaller reassembles and calls this insertion with or without indel realignment

Indel realigned reads that cross above HaplotypeCaller's MAPQ threshold alter the allelic and total depth counts for a variant site and can affect genotyping and genotype probability and quality scores as well as other annotation metrics.

HaplotypeCaller gains previously MAPQ10–19 reads now over the default MAPQ20 threshold of the HCMappingQualityFilter.

HaplotypeCaller and FreeBayes,

Longer short reads, deeper coverage and increased library complexity with PCR-free preparations give us higher quality sequence data.

We still recommend indel realignment for these legacy workflows

some “padding” to the intervals in order to include the flanking regions (typically ~100 bp). No need to modify your target list; you can have the GATK engine do it for you automatically using the interval padding argument.

This excludes off-target sequences and sequences that may be poorly mapped, which have a higher error rate. Including them could lead to a skewed model and bad recalibration.

There are even some analyses which should be restricted to the capture targets because failing to do so can lead to suboptimal results.

you could also provide a list of "bad" intervals with -XL, which does the exact opposite of -L:

Unless your database of variation is so poor and/or variation so common in your organism that most of your mismatches are real snps, you should always perform recalibration on your bam file.

Custom formula is: =MOD(INT((ROW(1:1)-1)/4),2)=0    Color: Gray   Range:  A1:Z1000 (or whole sheet...Ctrl+A)

while those that had been moved to 25 degrees had largely lost the methylation tags. Importantly, they still maintained this reduced histone methylation when moved back to the cooler temperature, suggesting that it is playing an important role in locking the memory into the transgenes.

Worms are very short-lived, so perhaps they are transmitting memories of past conditions to help their descendants predict what their environment might be like in the future,

cd /usr/local/RepeatMasker perl ./configure

human, mouse, zebrafish, fruit fly, and nematode

RepeatMasker was developed using TRF version 4.0.4

For RMBlast ( NCBI Blast modified for use with RepeatMasker/RepeatModeler )

The Administration could also exercise its regulatory authority—most potently, to direct the Centers for Medicare and Medicaid Services (CMS) to allow reimbursement for molecular profiling of cancers

assuming the rate of false positives and false negatives are the same for all mutations

cells that are hetero- or homozygous for a given mutation.

While it seems like such multiple mutations should be unlikely, computational biologist Niko Beerenwinkel of ETH Zurich in Basel, Switzerland, recently posted a preprint to bioRxiv suggesting that recurrent mutations can and do occur, and not infrequently (bioRxiv, doi:10.1101/094722, 2016).

So I quit. While my wife worked, I stayed home with our daughter Kriya, who was born in 1993, and became a house husband. I really didn't know what I wanted to do

I felt like every good result I had provided justification for a hundred lousy papers to follow, and that was a waste of people's time and taxpayers' money.

I tried the technology everywhere I could think of. Sometimes it worked and sometimes it didn't, but the papers came quick. In 1992, we applied it to data storage - at one time we held the world record for storage density - and in the following year I demonstrated super-resolution fluorescence imaging of cells for the first time.

I would come into work at 4:30 in the morning, and if I saw Harald's car, I would put my hand on the hood to find out if the engine was still warm. He did exactly the same thing. We were both really competitive, but we played tennis every morning and ate dinner together every night. We were best friends and still are.

I'm also lucky in that I have a second chance to be a better husband and father. While I'm close with Kriya and Ravi, one of my regrets is that I didn't spend more time with them when they were growing up

My group at Janelia has never been larger than five postdocs, and has averaged three

Everywhere I've been, I've been able to focus 100% on my work - I've never written a grant in my life. I doubt I would have been as successful in a more traditional academic career path.

That brought me back full circle to the optical lattice theory I had published in 2005.

The field was getting crowded, and I've always found it most productive to go where the people aren't. It was time to do something new.

Marty had told Gerry that I was interested in that "biological Bell Labs," HHMI's new Janelia Farm Research Campus. The campus wasn't built yet, but I was invited to interview in a little building off site in August, and was on the payroll in October 2005.

By early 2006, we had 20 nanometer resolution images of actin filaments, focal adhesions, mitochondria, and lysosomes. We submitted the work to Science in March, and it was published that August, after a lengthy fight with a reviewer who demanded correlative EM data, and then pushed for rejection even after we supplied it.

Harald and I built the first PALM microscope in his living room in La Jolla (Figure 6). We were both unemployed, but Harald had some of his equipment from Bell. We pulled that out of storage, and each put in $25,000 to cover everything else we needed. We worked hard, and in September shipped all the parts to rebuild the microscope in the darkroom of Jennifer's lab at the NIH. The first time we put a cover slip coated with molecules into the microscope and turned on the photoactivating light, the first subset popped up and we knew we had it.

Harald and I didn't know any biology, so we needed help.

Mike had one of the biggest libraries of fluorescent protein fusions in the world, and that's where we learned about photoactivatable fluorescent proteins. In the Tallahassee airport on our way home, it became obvious to Harald and me that this was the missing link for the idea that I had pitched after I left Bell

He invited me to present the idea to the biology department there in April 2005. Marty Chalfie was one of my hosts during that visit, and he turned to me in the cab on the way to dinner and said, "It sounds like you really believe in this idea. How are you going to get back in the lab?" I said, "I have no idea, but I read in Physics Today that there's a guy named Gerry Rubin who wants to make a biological Bell Labs," and we left it at that.

you know something so well that you love it and hate it and it's part of you. Within three months, I grokked diffraction and light and formation of foci.

I wanted to take advantage of GFP to do live cell imaging, but my physics knowledge had atrophied. So I pulled out my old textbooks and started redoing old homework problems. I was really motivated to understand it this time around, because I figured this was my last chance to make a scientific career.

I started reading the scientific literature again, and quickly came across Marty Chalfie's paper on green fluorescent protein, which he had published in 1994 as I was leaving Bell. It was like a religious revelation to me.

I reconnected with Harald, who had also gone into industry in San Diego, but wasn't completely satisfied.

So in 2002 I quit. That was probably the hardest part of my life. I had pissed away my academic career and I had pissed away my backup plan of working for my Dad. Once again, I didn't have any idea what I wanted to do. Fortunately, with money in the bank, I had some time to think of a solution.

Michigan in 1997 and set down roots

When Kriya was three, and started speaking with a Jersey accent, I knew we had to get out of New Jersey.

During my third year, I stopped just trying one thing after another, and started thinking like a physicist about why things weren't working.

Two years in, I wrote in my self-evaluation that if I didn't have a breakthrough in the next year, they wouldn't need to fire me because I would quit.

I knew next to nothing about semiconductor physics, but Horst thought near-field was a really cool idea and it could go places. He wanted to hire me, even though what I was doing was completely outside of what everyone else in the department was doing. Except for one guy - Harald Hess, who I also met during that visit. Harald had built a low temperature scanning tunneling microscope to study superconductors, and he and I hit it off immediately.

I built this crazy, elaborate, expensive microscope that kind of worked (Figure 2). I never looked at much beyond test patterns, but it was enough to prove that the idea was valid.

the vulnerability of cancer cells that have lost one copy of an essential gene might create a therapeutic window