No abstract

BackgroundLong-read nanopore sequencing technology is of particular significance for taxonomic identification at or below the species level. For many environmental samples, the total extractable DNA is far below the current input requirements of nanopore sequencing, preventing “sample to sequence” metagenomics from low-biomass or recalcitrant samples.ResultsHere we address this problem by employing carrier sequencing, a method to sequence low-input DNA by preparing the target DNA with a genomic carrier to achieve ideal library preparation and sequencing stoichiometry without amplification. We then use CarrierSeq, a sequence analysis workflow to identify the low-input target reads from the genomic carrier. We tested CarrierSeq experimentally by sequencing from a combination of 0.2 ng Bacillus subtilis ATCC 6633 DNA in a background of 1000 ng Enterobacteria phage λ DNA. After filtering of carrier, low quality, and low complexity reads, we detected target reads (B. subtilis), contamination reads, and “high quality noise reads” (HQNRs) not mapping to the carrier, target or known lab contaminants. These reads appear to be artifacts of the nanopore sequencing process as they are associated with specific channels (pores).ConclusionBy treating sequencing as a Poisson arrival process, we implement a statistical test to reject data from channels dominated by HQNRs while retaining low-input target reads.

Recent studies regarding the origin of life and Mars-Earth meteorite transfer simulations suggest that biological informational polymers, such as nucleic acids (DNA and RNA), have the potential to provide unambiguous evidence of life on Mars. To this end, we are developing a metagenomicsbased life-detection instrument which integrates nucleic acid extraction and nanopore sequencing:The Search for Extra-Terrestrial Genomes (SETG). Our goal is to isolate and sequence nucleic acids from extant or preserved life on Mars in order to determine if a particular genetic sequence (1) is distantly-related to life on Earth indicating a shared-ancestry due to lithological exchange, or (2) is unrelated to life on Earth suggesting a convergent origin of life on Mars. In this study, we validate prior work on nucleic acid extraction from cells deposited in Mars analog soils down to microbial concentrations observed in the driest and coldest regions on Earth. In addition, we report low-input nanopore sequencing results equivalent to 1 ppb life-detection sensitivity achieved by employing carrier sequencing, a method of sequencing sub-nanogram DNA in the background of a genomic carrier.

Recent studies regarding the origins of life and Mars-Earth meteorite transfer simulations suggest that biological informational polymers, such as nucleic acids (DNA and RNA), have the potential to provide unambiguous evidence of life on Mars. To this end, we are developing a metagenomics-based life-detection instrument which integrates nucleic acid extraction and nanopore sequencing: the Search for Extra-Terrestrial Genomes (SETG). Our goal is to isolate and sequence nucleic acids from extant or preserved life on Mars in order to determine if a particular genetic sequence (1) is distantly related to life on Earth, indicating a shared ancestry due to lithological exchange, or (2) is unrelated to life on Earth, suggesting a convergent origins of life on Mars. In this study, we validate prior work on nucleic acid extraction from cells deposited in Mars analog soils down to microbial concentrations ( i.e., 10 4 cells in 50 mg of soil) observed in the driest and coldest regions on Earth. In addition, we report low-input nanopore sequencing results from 2 pg of purified Bacillus subtilis spore DNA simulating ideal extraction yields equivalent to 1 ppb life-detection sensitivity. We achieve this by employing carrier sequencing, a method of sequencing sub-nanogram DNA in the background of a genomic carrier. After filtering of carrier, low-quality, and low-complexity reads we detected 5 B. subtilis reads, 18 contamination reads (including Homo sapiens ), and 6 high-quality noise reads believed to be sequencing artifacts.

24Motivation: Long-read nanopore sequencing technology is of particular significance for 25 taxonomic identification at or below the species level. For many environmental samples, the total 26 extractable DNA is far below the current input requirements of nanopore sequencing, preventing 27 "sample to sequence" metagenomics from low-biomass or recalcitrant samples. 28Results: Here we address this problem by employing carrier sequencing, a method to sequence 29 low-input DNA by preparing the target DNA with a genomic carrier to achieve ideal library 30 preparation and sequencing stoichiometry without amplification. We then use CarrierSeq, a 31 sequence analysis workflow to identify the low-input target reads from the genomic carrier. We 32 tested CarrierSeq experimentally by sequencing from a combination of 0.2 ng Bacillus subtilis 33 ATCC 6633 DNA in a background of 1 µg Enterobacteria phage λ DNA. After filtering of carrier, 34 low quality, and low complexity reads, we detected target reads (B. subtilis), contamination reads, 35and "high quality noise reads" (HQNRs) not mapping to the carrier, target or known lab 36