One of the limitations of the family trio work was that the bioinformatics pipelines were different between our samples and our kids’ samples. To fix this limitation, I had to “reconstitute” the original FASTQ files from the BAM file provided by Illumina and then re-run all our data through the same pipeline. (Note: To my knowledge, UYG no longer provides BAM files as part of this program.)
You can create FASTQ files from your BAM file by using Picard, a set of Java-based command line tools for manipulating high-throughput sequencing (HTS) data in formats such as SAM/BAM/CRAM and VCF.
For reasons that escape me now, I first ran Picard using an AWS t1.micro instance.
After 3 attempts–watching Picard fail after running for 3 days each time–and creating thousands of temp files in the process, I learned the hard way that Picard requires more than 613 MBytes of memory. This time, I used a c4.2xlarge instance (4 cores, 16 GBytes of memory), which worked. It appears that 16 GBytes is about the minimum amount of memory to get the job done.
Step 1. Is your BAM file sorted?
Before creating FASTQ files, make sure your BAM file is sorted so that your genome coordinates are in order. One of the ways to do this is with samtools, a suite of programs for interacting with HTS data. Here are the commands I used to install it. You can check whether or not your BAM file is sorted by using this command:
samtools stats YourFile.bam | grep "is sorted:"
# "is sorted: 1" = Yes, your BAM file is sorted.
# "is sorted: 0" = No, your BAM file is not sorted.
If your BAM file requires sorting, use this command (or something close to it):
# Type "samtools sort --help" for a description of this command
samtools sort -n -@ 2 -m 2560M InputFile.bam -o ./OutputFile.sorted.bam
# Check for existence of Read Groups (@RG)
samtools view -H InputFile.bam | grep '^@RG'
Step 2. Run Picard
Get Java and the picard.jar file. Run this command, but keep in mind that the options below are for a BAM file created on an Illumina HiSeq sequencer:
Using the c4.2xlarge instance, I ran Picard in 3 hours to create the FASTQ files shown below. In addition, creating compressed (gzip) versions of the files required another 8.5 hours of compute time. With an on-demand price of about $0.40 per hour, creating compressed FASTQ files cost approximately $4.60 USD on AWS.
I launched my first cloud server literally while in the clouds in May 2014. Cloud computing has changed so much, it’s unbelievable. Back then, I had to patch the Linux kernel by hand so that the ftp server would work on AWS. Today, uploading your genome using Amazon’s command line interface (CLI) to an AWS S3 storage bucket is relatively easy. Understandably, Amazon makes it challenging (but doable) to make your storage publicly available. I used the Apache Web Server and s3fs to share this information.
s3fs allows allows you to mount an S3 bucket via FUSE. s3fs preserves the native object format for files, allowing use of other tools like AWS CLI. Again, your commands may vary depending on your flavor of Linux. Here are the commands I used to install s3fs.
Great news! Six months have passed since Kimberly finished radiation therapy for breast cancer. Today, she had a follow-up diagnostic mammogram that confirmed she is cancer-free! She will continue to be monitored over the next 5 years, but our big worries are behind us. Incidentally, we learned about a useful website during our journey, cancersurvivalrates.com that gave us a much better picture of survival rates.
Hereditary cancer screening
Let’s finish by returning to the variant in the APC gene that we found during expanded genetic testing and wrap-up this series.
During genetic testing, our genetic counselor ordered an additional gene panel to screen for other cancers due to Kimberly’s family history. As I mentioned earlier, our insurance company denied all of our genetic testing claims, saying that the expanded panel was not related to her breast cancer. Nevertheless, the information that we received was worth the $250 out-of-pocket expense. Given the lack of reimbursement, reasonable costs for clinical genetic testing will ultimately drive most of it to be physician-ordered but privately paid. Just be sure to get your data!
So, what did we learn?
As we know from autosomal dominant inheritance, a person affected by an autosomal dominant disorder has a 50 percent chance of passing the mutated gene to each child. And sure enough, we saw the APC gene variant in 1 of our 2 adult-aged children; the other child does not carry it. We know this because we have whole genome sequences for everyone in our family. Here’s what Kimberly’s genetic code looks like at this location:
It turns out that this variant increases the risk of colorectal cancer from 5% (found in the general population) to 10% (in the population with this variant). So, the child with the variant should have a colonoscopy at age 40 (earlier than usual) and follow-up colonoscopies every 5 years after that. If you have a APC gene variant, talk to a genetic counselor–and show them some love! Note: This blog is not intended to replace advice from a medical professional.
Before publishing this story, we had a family meeting to discuss Mom’s cancer-free diagnosis, as well as the APC variant that one of them carries. All of us agreed to share this information with hopes that it will assist others.
Along the way, we learned that knowledge gave us the strength to move forward. I also have newfound appreciation for my wife, whose bravery knows no bounds.
One month after surgery, Kimberly began radiation therapy, which is designed to reduce the recurrence of breast cancer after surgery by more than half. We met with a radiation oncologist and developed a 15-visit treatment plan. The cost of Kimberly’s radiation therapy was about $25,000, and fortunately our health insurance covered about 90%.
Radiation therapy and genetics have a curious relationship. The basic idea behind radiotherapy is to induce double-strand breaks in DNA with ionizing radiation. Although radiation damages both normal cells and cancer cells, most normal cells repair themselves, while cancer cells do not. Therapy is given in daily doses to allow the DNA in healthy cells to recover between visits.
External beam radiotherapy based on linear accelerators has been available since the early 1950s, and machines like the Varian Clinac above deliver a shaped beam of high-energy x-rays to a precisely targeted area. In Kimberly’s case, a surgeon had removed her tumor 1 month prior, so the target area was the breast where the surgery occurred–just in case a single errant cancer cell had wandered from the surgical site.
We made daily visits for several weeks and Kimberly tolerated the procedure well. On her right side she had what looked like a sunburn, a common side effect, that faded over the next month. We continued to have follow-up visits with both her medical and radiation oncologists.
A few days after finishing radiation therapy, we visited the Varian production plant in Palo Alto, California. It was fascinating to see the construction of these behemoth machines and learn more about their operation. (My favorite part was learning that the electron linear accelerator tube is tuned with a ball peen hammer.) As luck would have it, all of this activity occurred just 1 week before the COVID-19 shelter-in-place order hit the San Francisco Bay area in March 2020.
We spent the next 6 months not only sheltering-in-place, but also waiting for her follow-up mammogram to determine if radiation therapy was successful.
The day after Kimberly received her breast cancer diagnosis, we met with a board-certified genetic nurse, Frank. Before our visit, we completed a form that Frank used to create a family pedigree. The list below is not a pedigree, but it shows the history of cancer in Kimberly’s family. We removed kinship for privacy:
Breast cancer (maternal side: 1, paternal side: 1)
Lung/bone cancer (paternal: 3)
Cervical cancer (maternal: 1)
Colon cancer (paternal: 1)
Liver cancer (paternal: 2)
Kidney/bone cancer (paternal: 1)
Esophageal cancer (paternal: 1)
It turns out that 5-10% of cancer is inherited. People who carry hereditary mutations do not necessarily get cancer, but their lifetime risk is higher than average. Genetic counselors use pedigree charts to visualize family history and evaluate when genetic testing adds diagnostic value. Kimberly’s family history met lab guidelines for further evaluation, so Frank ordered a gene panel from a nearby lab, Invitae. The blood test was ordered stat, and we received our results six business days later. Treatment plans can change based on genetic results, so we were grateful to receive results before her surgery, which was now scheduled.
We returned to Frank’s office and first learned that she does not carry mutations in 9 genes known to influence the risk of breast cancer: ATM, BRCA1, BRCA2, CDH1, CHEK2, PALB2, PTEN, STK11, and ΤΡ53. Phew! Invitae also provides free hereditary cancer testing to breast cancer patients at no additional charge (as long as you order the expanded panel within 90 days of the original test), so Frank ordered the expanded panel. Given that Kimberly has a family history of other hereditary cancers, we welcomed a broader genetic search. The results could be meaningful not only to us, but also to other living relatives. Oddly, our insurance company rejected all of our genetic testing claims because the resubmission was not related to her breast cancer diagnosis. I discussed the situation with Invitae and they were very accommodating–our total out-of-pocket cost was $250. I am still mad at our insurance company, but that’s a rant for another day.
Although she did not have any mutations related to breast cancer, Kimberly’s expanded genetic testing revealed a point mutation in the APC gene, which is known to increase the risk of colorectal cancer. People with this variant are generally counseled to have their first colonoscopy at age 40 (she did that) and follow-up colonoscopies every 5 years (coming up). Since the APC I1307K variant is autosomal dominant, close relatives such as siblings and children have a 1 in 2 chance of inheriting an APC mutation. We called Kimberly’s sister and shared our findings, part of a cascade testing strategy. We also have our kid’s whole genome sequences, which will let us check for APC mutations directly. We will return to that search in part 4 of this series.
We left Frank’s office and developed a treatment plan with Kimberly’s surgeon and medical oncologist a few days later. The plan included surgery (lumpectomy) followed by radiation therapy. Surgery was successful, as you can see in the before and after images below. (Special thanks to the Horos Project for the open source DICOM viewer.)
Kimberly received her diagnosis the day after Thanksgiving. In the 18 days that followed, we had 16 medical appointments that took us from diagnostic mammogram to surgery. With surgery behind us, Christmas was now six days away. We spent a quiet holiday with the kids.
We began 2020 hopeful, knowing that her type 1A tumor had been successfully removed by her surgeon. We were also much more knowledgeable about hereditary cancer risks due to Frank’s counseling.
One month later, Kimberly would begin radiation therapy to dramatically decrease her chance of recurrence.
It was the day after Thanksgiving. My wife Kimberly was talking with a nurse about the results from a biopsy performed 2 days earlier. She hung up her mobile phone and burst into tears. Kimberly received the call while we were exiting the gates of Leavenworth National Cemetary in Kansas, where we had just laid my mother-in-law Barbara to rest with her husband, Gilbert. Our kids were in the back seat and did not really know what was going on, but they guessed that mom had cancer.
The week prior, Kimberly had a diagnostic mammogram, and the radiologist told us in person that Kimberly had a suspicious lesion in her right breast (larger than a peppercorn, smaller than a pea) and recommended a biopsy. Luckily, a biopsy appointment was available the day before Thanksgiving, and we took it even though we were flying to Kansas City the next day. We asked the care coordinator to call us as soon as she had preliminary pathology results, and she did. Our family flew home to the San Francisco Bay area on Sunday.
On Monday, Kimberly and I visited the medical oncology department of a nearby clinic. The nurse said that Kimberly had invasive ductal carcinoma. Surprisingly, the rest of the visit did not turn into that dull surreal buzz that often accompanies bad news and drowns out everything else. In our case, years of being in rooms like this one discussing the needs of our exceptional children proved immensely useful. I took notes and Kimberly asked incisive questions about treatment options, radiation therapy, and genetic counseling. The nurse patched-in our long time family physician over the phone, and his presence was very assuring. It was a brief respite from what would become an overwhelming 3 month journey–the first 2-3 weeks especially so. We learned about a bewildering array of cancer treatment options, visited competing medical facilities, and evaluated new doctors.
Those odds were not good, and I had multiple panic attacks over the next few weeks at the thought of losing my wife. “Hang in there. Moment by moment,” a friend texted to me. I read that message over and over, hanging on.
$1,500. That’s the amount of money I have spent over the past 5 years to store our family’s whole genome sequence (WGS) data. For $299 per person in 2020, I could sequence all of us again at 30x coverage, get the same data files, and spend less money. In 2015, I wrote about posting my WGS data to DNAnexus. Last month (July 2020), I moved all of our data to Amazon (AWS) S3 storage. In this post, I explain why.
Nevertheless, I recently moved my WGS data to Amazon S3 due to storage costs and a lack of price transparency.
I’ve learned that most of the work that I want to do can be done with VCF files. Yes, there are times when I want to look at BAM files, but moving those files to lower-cost storage makes sense. DNAnexus introduced a Glacier-based archiving service in 2019 to support those operations, although I did not use it. My BAM file is 73 GBytes, which represents about 93% of the 79 GBytes for my WGS data (no FASTQ data). If I deeply archive BAM and FASTQ data (329 GBytes total), I can reduce the amount of higher-cost storage by 98%. The cost comparison for a single genome with FASTQ files looks roughly like this:
Storage cost on DNAnexus: (329 GBytes * $0.03 per GB-month [everything]) = $9.87 per month
Storage cost on AWS: (7 GBytes * $0.0125 per GB-month [VCF]) + (322 GBytes * $0.00099 per GB-month [everything else]) = $0.41 per month
Overall, I can reduce my monthly storage costs by over 95% by using lower-cost storage tiers on AWS (see Table 1 below). Again, the comparison is apples-to-oranges because I did not use DNAnexus’ archiving service, mostly because it required a separate license to activate. Using Amazon S3, our monthly WGS storage costs will decrease from $24 per month to less than $1 per month.
Lack of price transparency
If we compare AWS’ S3 storage price from 5 years ago to DNAnexus’, we find that the storage markup was 35% over the S3 list price. It turns out that Amazon decreased its S3 storage price over the past 5 years, which led DNAnexus to drop their storage price to the current $0.03 per GB-month, still at a 35% markup. (For comparison, on demand GPU- or FPGA-based compute cycles (Amazon EC2) are marked-up over 100%.)
I do not fault DNAnexus for marking-up AWS pricing–they are a business and provide value beyond storage and compute cycles. However, you will not find any pricing information on the DNAnexus website. In addition to storage costs, add-ons like archiving and GxP regulatory compliance require separate licenses that are not disclosed when signing-up. Presumably, the company’s professional services team assists with these onboarding activities.
How to move your data from DNAnexus to AWS
So, having made the decision to move my WGS data to AWS, how did I do it?
On the DNAnexus platform, I used AWS S3 Exporter, a company-provided tool to upload data to an AWS S3 bucket (DNAnexus account required). You can invoke the exporter using either their SDK (dx-toolkit) or an online wizard–both methods work great. The DNAnexus documentation for the exporter tool is a little out-of-date, so here is the updated AWS IAM policy file to make your transfers work with verification:
Another improvement: You can transfer your data from one S3 instance to another (DNAnexus to AWS) at the rate of 250 GBytes per hour, including verification. Five years ago, the transfer speed was 10 GBytes per hour!
One final gotcha
One thing that has not changed in 5 years is the “data transfer out” fee. Amazon’s fee is $0.09 per GByte and DNAnexus’ fee is $0.13 per GByte. This fee is an understandable disincentive to keep you from moving your data around too much. In my case, moving our family’s WGS data to AWS will add over $100 to the final bill. It’s a little like losing all your money at baccarat and then finding out that you still owe the banque a commission before you leave the table. Not a big deal when you are a family, but when you are the UK Biobank expecting to grow to 15 petabytes over the next 5 years…well, you get the idea.
For the money, take a look at upstart competitors like Basepair or ixLayer.
You may know this painting, one of over two dozen haystack paintings (actually, stacks of wheat) that Claude Monet produced at various times and seasons. What if we hid a needle one of them? Could we find it? In genetics, we often say that finding a genetic variant is like finding a needle in a haystack. But how big is the needle? How big is the haystack? Who believes this stuff?
Francis Collins, NIH director and leader of the Human Genome Project, is a clear believer. In a recent PBS series by Ken Burns, The Gene: An Intimate History, Dr Collins said that finding a misspelling in a gene that causes a particular disease is “a needle in a haystack.“
So, in honor of April 25th, National DNA Day, which commemorates the discovery of DNA’s double helix in 1953 and the publication of the first draft of the human genome in 2003, I embarked on a curious journey to answer these questions. My approach would be to determine the volume of a needle and then figure out how large to make the haystack to see if the analogy holds.
What is the volume of a needle?
How do you calculate the volume of a needle? It is useful to start with some real needles, and it turned out that I had a small box of 30 assorted needles–here’s a photo of them:
Needles come in all kinds of lengths, but I was looking for an “average” needle, so I made a rough list of their lengths and came up with this:
Average needle length: sum(5 x 30 mm, 10 x 35 mm, 11 x 40 mm, 1 x 45 mm, 1 x 50 mm, 1 x 55 mm, 1 x 60 mm) / 30 = 37 mm or 1.4 inches
Volume of a needle: displacement method
Next, I had to figure out the volume (V) of a needle. My first approach was to drop the needles into a graduated cylinder filled with water and then measure the displacement.
It turns out that 30 needles displaced 0.5 ml of water, so we divide by 30 to get the average volume of the needles in my sample.
V = 0.5 mm / 30 = 0.016 ml or about 0.02 ml per needle
Can we find a different method to measure the volume of the needle and compare results?
Volume of a needle: small cylinder
Another way to approximate the volume of the needle is to view it as a small cylinder using the formula: V = pi * r^2 * height. Since we are looking for volume (V), we want to express our measurements in centimeters to end up with cubic centimeters (cc).
V = pi * (0.075 cm / 2)^2 * 3.7 cm = 0.016 cc, or about 0.02 cc
Since 1 ml = 1 cc, we can say that the volume of our needle is 0.02 ml, which is spot on with our previous measurement. Note: It is rare in science that your numbers match exactly, but we’ll take the win and start building our haystack.
Building a genetic haystack
Our next task is to build a haystack that approximates the size of the human genome, where each A-T or C-G base pair in the human genome is the size of a needle. The Human Genome Project pegs that number around 3 billion base pairs within one copy of a single genome.
We build our genetic haystack by defining it as a volume having 3 billion opportunities to find a 0.02 ml object, or volume (V) equals 3,000,000,000 * 0.02 ml.
V = (3*10^9 * 0.02 ml) = 60*10^6 ml or 60,000,000 ml
Since 1 ml = 1 cubic centimeter (cc), the volume can also be stated as 60,000,000 cubic centimeters or 60*10^6 cc. Finally, 1 liter = 1000 ml, so the volume in liters equals 60,000,000 ml * (1 liter / 1000 ml).
V (haystack) = (60*10^6 ml) * (1 liter / 1000 ml) = 60*10^3 liters or 60,000 liters
We now know how much space it occupies, but what does it look like? For example, how tall is our haystack?
Shape of a genetic haystack: approximation with a hemisphere
Like the needle, we can approximate the shape of a haystack to something easy to calculate, like the volume of hemisphere in cubic centimeters: V = 2/3 * pi * r^3.
V (hemisphere) = 60*10^6 cc = 2/3 * pi * r^3
Solving for the radius (r), we get r = 306 cm. Since 1 m = 100 cm, the height (radius) of the hemisphere equals 306 cm * (1 m / 100 cm).
r (hemisphere) = 306 cm * (1 m / 100 cm) = about 3 meters or 10 feet tall
So, we have a rough idea of what an idealized haystack looks like. It is 3 meters tall and it is shaped like a hemisphere. But how accurate is our measurement? Can we do better? Has anyone studied haystack modeling? Indeed, someone has and his name is W.H. Hosterman.
Shape of a genetic haystack: using haystack modeling
In 1931, W.H. Hosterman from the U.S. Department of Agriculture published an extensive technical bulletin “for the purpose of determining the volume and tonnage of hay.” Hosterman and his USDA colleagues measured over 2,600 haystacks across 10 states and presented results for both square and round haystacks. For round haystacks, Hosterman derived this formula: V = ((0.04 * Over) – (0.012 * C)) * C^2, where C is the circumference of the haystack, and Over is the measurement of the circumference of the stack over the top. We can compare results with the hemisphere by selecting equivalent values from Table 7 in Hosterman’s paper. (We will momentarily switch to imperial units to make use of the constants in the formula.)
The observed height of Hosterman’s haystacks are slightly taller than wide, but their sloped tops make the volume of the “true” haystack (58,333 liters) a little less than our original estimate of a 3-meter-tall, 60,000-liter haystack. When we compare the volume using Hosterman’s formula to the volume of the hemisphere, we see that they differ by 1,667 liters, or about 3%. Close enough!
Genetics is like finding a needle in a haystack
We have two comparisons that match closely. So, what does that haystack look like?
It turns out that Romanian haystacks are about 3 meters tall, so finding a misspelling in a gene (a genetic variant) is indeed like finding a needle in a haystack.
Who looks for needles in haystacks?
If genetics truly is like finding a needle in a haystack, what kinds of people do this? Well, let’s go back to Francis Collins.
In 1993, Collins published a paper describing the genetics of cystic fibrosis, a disease that required finding “a needle in a haystack.” Today, we have found needles from thousands of genetic haystacks. Some of these needles lead to clues about rare diseases, which affect more than 400 million people worldwide.
In real life, you can find a needle in a haystack, too. In 2004, performance artist Sven Sachsalber found a needle in a haystack after hunting for it for 24 hours. If we could get computers to solve diseases at that speed, we would happily be out of work.
Today I joined All of Us, a research community of one million people to lead the way for individualized prevention, treatment, and care for, well, all of us. This project was previously known as the Precision Medicine Initiative.
Many of you know that our family has used whole genome sequencing to look for clues in our daughter’s autism. This blog shares that journey. I have also published peer-reviewed papers to explore the reasons why people share personal health information. Through this research, I am convinced that information sharing will contribute to a learning healthcare system to improve care and lower costs.
It just takes people like you and me to #JoinAllofUs and lead by example.
In 2006, a Scientific American article written by George Church, “Genomics for All,” rekindled my interest in genomics. I went back to school in 2009 to contemplate the business of genomic medicine, and celebrated my MBA by writing a Wikipedia entry for the word, “Exome.” I was hooked.
Along the way, I realized that medical imaging and genomics are highly complementary: genomics informs or identifies conditions, and radiology localizes them. Sarah-Jane Dawson pointed this out at a Future of Genomic Medicine conference in 2014.