Scott Edmunds

[![](http://gigasciencejournal.com/blog/wp-content/uploads/2013/04/396093_122813224529754_274194545_n-300x225.jpg){.alignleft
.size-medium .wp-image-484 loading="lazy" decoding="async" width="300"
height="225"
srcset="http://gigasciencejournal.com/blog/wp-content/uploads/2013/04/396093_122813224529754_274194545_n-300x225.jpg 300w, http://gigasciencejournal.com/blog/wp-content/uploads/2013/04/396093_122813224529754_274194545_n-768x576.jpg 768w, http://gigasciencejournal.com/blog/wp-content/uploads/2013/04/396093_122813224529754_274194545_n.jpg 960w"
sizes="(max-width: 300px) 100vw, 300px"}](http://gigasciencejournal.com/blog/wp-content/uploads/2013/04/396093_122813224529754_274194545_n.jpg)Following
from our [previous blog
posting](http://blogs.biomedcentral.com/gigablog/2013/03/27/new-in-gigascience-the-squishome/ "Squishome GigaBlog posting"){target="_blank"
rel="noopener"}, here we profile and interview Dr Xin Zhou, lead author
of our recent "squishome" insect goo metabarcoding
[paper](http://dx.doi.org/10.1186/2047-217X-2-4 "Insect goo paper"){target="_blank"
rel="noopener"}. This NGS (next generation sequencing)-based work has
already generated a lot of interest (see [this
write-up](http://www.wired.co.uk/news/archive/2013-03/27/insect-dna "Wired squishome write-up"){target="_blank"
rel="noopener"} in Wired and this [blog
posting](http://dna-barcoding.blogspot.hk/2013/03/ultra-deep-sequencing.html? "DNA metabarcoding blog"){target="_blank"
rel="noopener"} for nice examples), and here Dr Zhou gives more insight
into the potential for the technique in studying biodiversity, as well
as some of the quirky findings his team made validating the technique
behind their laboratory in China.

Dr Xin Zhou is Director of the Environmental Genomics research group at
[BGI](http://www.genomics.cn/en/index "BGI Shenzhen website"){target="_blank"
rel="noopener"}, and Director of the Bio-resource Bank of the [China
National
GeneBank](http://www.nationalgenebank.org/en/index.html "China National Genebank homepage"){target="_blank"
rel="noopener"}. A biodiversity expert and Entomologist by training, Dr
Zhou carried out his postgraduate studies and postdoctoral training at
Rutgers University and University of Guelph, managing barcoding projects
for the [International Barcode of
Life](http://www.ibol.org/ "iBOL database"){target="_blank"
rel="noopener"}. From assembling and curating barcode reference
libraries for a number of aquatic insect groups, his work has moved to
the development of sequencing based analytical pipelines for bulk insect
samples at Guelph, and since moving to China in October 2010, at BGI.

**How is this method of PCR-free metabarcoding an improvement on
previous techniques?**

XZ: In PCR-based metabarcoding approaches, various primer sets are used
to amplify target DNA fragments, which almost always introduce taxonomic
biases, such that some organisms are easier to be detected while others
are constantly missing or under-represented. This artificial bias poses
a serious problem to all biodiversity studies where species composition
is of a concern. Our work is the first of its kind that shows analyzing
natural biodiversity samples doesn't have to rely on PCR therefore
bypassing the primer issue. In addition, our work demonstrates that the
PCR-free pipeline may potentially reveal species abundance from the
mixed arthropod sample, providing yet another piece of crucial
information to ecologists alike.

**What does this NGS-based technology bring to it over just manually
barcoding the collected samples?**

XZ: Significantly reduced time and labor in sample processing as well as
overall cost to analyze bulk samples.

**What can you actually do with this the data, and what potential new
applications does this technique enable? If you can say, what are you
planning to do with this technique next?**

XZ: This paper is a proof of concept demonstrating that natural bulk
samples can be analyzed using NGS without having to relying on PCR
amplification. This is the first step towards empirical applications of
the new methodology in ecological and biodiversity related researches.
We demonstrate that this new pipeline CAN work while a few technical
issues can be improved for its wide implementation, such as in
mitochondrial enrichment and tissue preservation. While trying to
improve these technical details, we plan on increasing diversity scales
of arthropod samples by analyzing those collected in tropical regions
and arrays of insect samples collected from real-world ecological
sampling designs.

[![](http://gigasciencejournal.com/blog/wp-content/uploads/2013/04/IMG_0100_111-300x200.jpg){.alignright
.size-medium .wp-image-483 loading="lazy" decoding="async" width="300"
height="200"
srcset="http://gigasciencejournal.com/blog/wp-content/uploads/2013/04/IMG_0100_111-300x200.jpg 300w, http://gigasciencejournal.com/blog/wp-content/uploads/2013/04/IMG_0100_111-768x512.jpg 768w, http://gigasciencejournal.com/blog/wp-content/uploads/2013/04/IMG_0100_111-1024x683.jpg 1024w"
sizes="(max-width: 300px) 100vw, 300px"}](http://gigasciencejournal.com/blog/wp-content/uploads/2013/04/IMG_0100_111.jpg)**Why
did you collect the specimens you validated it with behind your lab at
BGI \[based in the large urban city of Shenzhen\], and were you
surprised by the number of specimens you detected?**

XZ: This was an advantage working in a subtropical region where
biological samples are relatively easy to obtain. Although the sampling
was not comprehensive in terms of intensity of traps and number of
species, we were surprised to see what we managed to collect in the
middle of a community township. The 2 sampling sites were very close to
each other yet there were merely \~10% of the total species being shared
between them. Also, the fact that only very few of our barcoded
specimens received a sequence match from the [Barcode of Life Data
Systems](http://www.boldsystems.org/ "iBOL database"), the world's
largest barcode reference database, suggests that much of China's
arthropod fauna still remains as a mystery, at least from a molecular
aspect. On top of all that, we thought it would be an interesting idea
to present BGI's headquarters in a scientific publication for the first
time, with its GPS coordinates recorded in a meta-database.

**Does this example say anything useful about the biodiversity in
Shenzhen and the area around the BGI HQ?**

XZ: as stated above, although the 2 arthropod bulk samples represent
typical fauna of a secondary forestry ecosystem in Southern China, the
overlap between samples was minimum and much of the community was poorly
understood both morphologically and molecularly. We believe there is an
urgent need to improve our knowledge on China's arthropod fauna. And we
will start from where we live. We have a plan to barcode and metabarcode
insects and plants of the Shenzhen municipal area.

**In the paper it was interesting you found a novel COI \[[cytochrome
oxidase subunit
I](http://en.wikipedia.org/wiki/DNA_barcoding#Mitochondrial_DNA "DNA barcoding wikipedia page"){target="_blank"
rel="noopener"} -- a common mitochondrial marker used in barcoding
studies\] from a
[Lepidoptera](http://en.wikipedia.org/wiki/Lepidoptera "Lepidoptera (butterflies and moths) species wikipedia"){target="_blank"
rel="noopener"} species not found in the reference library. Can you say
a little more on this example, and is this a potentially new species?**

XZ: Based on the quality of the nucleotide sequences and overall
coverage of the novel barcode, we tend to believe that this is a real
taxon that was somehow not detected in our morphological and barcode
examinations. However, given the protocols used in this work, it is not
possible to identify the exact source of this novel COI sequence. We
listed a few potential possible reasons, including gut content, small
residual tissues in the bulk sample, extracellular DNA etc. This novel
sequence doesn't get a sequence match in any existing barcode databases.
But this is not a big surprise as we know that Chinese insect species
are not well-sequenced. The ultra-deep sequencing capacity of the NGS
platforms opens up a new prospective where we are now capable of
revealing diversity of the even-smaller-things-that-run-the-world via
detecting their molecules. This would not have been possible if we had
to rely on the visual cues of these organisms. In some sense, the
contribution of NGS technology to biodiversity research is equivalent to
what microscopes did to microbiology.

**On that subject, what potential does this technique have to help
discover new species, and how much can you actually tell about them
using it?**

XZ: NGS technology creates an alternative way to analyze biodiversity
pattern and its temporal and spatial variations by detecting molecular
or genomic heterogeneity
([MOTUs](http://xyala.cap.ed.ac.uk/research/barcoding/motu_defined.html "MOTUs defined"){target="_blank"
rel="noopener"}) in bulk environmental samples. However, to make sense
of these molecular operational units, one would have to compare this
sequence information to well-curated sequence databases that are tied to
conventional biological species concepts. A good example of these
databases is the [Barcode of Life Data
Systems](http://www.barcodinglife.com/ "iBOL database"){target="_blank"
rel="noopener"}, where millions of barcode sequences are linked to
voucher specimens. My feeling is that the construction of sequence
reference databases will remain critical in future molecular/genomic
biodiversity research as it is a crucial step to provide linkages to the
classic school of organismal science. However, NGS cataloging of world
biodiversity can be performed in parallel. As long as meta-data are
maintained for the bulk samples, biodiversity can be registered as MOTUs
at first in a much accelerated fashion, and then be compared against
existing reference databases available at the time. Known and
(potentially) new species can be gradually revealed during this
procedure. As biodiversity registration can be significantly accelerated
using NGS, understanding biodiversity and especially interactions among
species will be a long-term endeavor.

**What are the implications for this technique in the growth of data
taxa in the databases? As it is more high-throughput does it have the
potential to massively increase the number of new entries?**

XZ: This
[PCR](http://en.wikipedia.org/wiki/PCR "PCR: polymerase chain reaction"){target="_blank"
rel="noopener"}-free approach can produce more accurate result in terms
of species composition for bulk biological samples.

In terms of impact in increasing data entries in the databases, I
believe this will be the future trend in biodiversity genomics. As the
emergence of new technologies and rapid reduction in costs, the research
community will be able to analyze much more biological samples in much
shortened periods of time. The outcome will be an improved understanding
of biodiversity changes based on consistent and standardized analysis
procedures and intensified sampling (in terms of numbers of sampling
sites across space and time and specimen numbers).

**Is there anything else that you want to tell me about the technique?**

XZ: the new PCR-free pipeline we created in this paper has further
potentials in terms of construction reference genomes, such as
mitochondrial and chloroplast genomes, in a much more economically
efficient way. Based on our findings in the present work, much of the
other mitochondrial genes of most of the insect species from the mixed
sample can also be assembled with a decent
[N50](http://en.wikipedia.org/wiki/N50_statistic "N50 statistic in sequencing"){target="_blank"
rel="noopener"} value. For instance, the largest scaffold we managed to
assemble from the insect soup was a moth representing almost the entire
length of its mitochondrial genome. This means that with some tweak of
the current pipeline, we would be able to sequence and assemble small
genomes for many different species in one shot. Having a comprehensive
reference library for mitochondrial genomes can solve many of the
difficult questions faced in the classic barcoding community, such as
primer designs for the standard barcode region for difficult groups,
e.g.,
*[Hymenoptera](http://en.wikipedia.org/wiki/Hymenoptera "Hymenoptera:  sawflies, wasps, bees and ants"){target="_blank"
rel="noopener"}*. Also this potential opens up the door to expanding
classic barcoding methods from the current single-molecule approach to
genomic screening.

**Further reading:**  
[1.](http://dx.doi.org/10.1186/2047-217X-2-4 "Insect goo paper"){target="_blank"
rel="noopener"} Zhou X; et al., Ultra-deep sequencing enables
high-fidelity recovery of biodiversity for bulk arthropod samples
without PCR amplification *GigaScience* 2013 **2**:4
[http://dx.doi.org/10.1186/2047-217X-2-4](http://dx.doi.org/10.1186/2047-217X-2-4 "Insect goo paper"){target="_blank"
rel="noopener"}

The post [Q&A with Xin Zhou, author of our insect "squishome"
paper.](http://gigasciencejournal.com/blog/qa-with-xin-zhou-author-of-our-insect-squishome-paper/){rel="nofollow"}
appeared first on
[GigaBlog](http://gigasciencejournal.com/blog){rel="nofollow"}.


Following from our previous blog posting, here we profile and interview Dr Xin Zhou, lead author of our recent “squishome” insect goo metabarcoding paper.

Messaggi di Rogue Scholar

Q&A with Xin Zhou, author of our insect “squishome” paper.